(12)
(19)
PATENT
AUSTRALIAN PATENT OFFICE
(11) Application No. AU 199888312 B2
(10) Patent No. 755822
(54)
Title
Agonist antibodies to the thrombopoietin receptor, and their therapeutic uses
(51) 7
International Patent Classification(s)
A61 K 039/395 C1 2N 01 5/1 0
C07K 01 6/28 C1 2N 01 5/62
C07K 01 7/00 C1 2N 01 5/85
C07K 019/00
(21)
Application No: 199888312
(22) Application Date: 1998.08.21
(87)
WIPONo: WO99/10494
(30)
Priority Data
(31)
Number (32) Date
08/918148 1997.08.25
(33) Country
US
(43)
(43)
(44)
Publication Date : 1999.03.16
Publication Journal Date : 1999.05.13
Accepted Journal Date : 2002.12.19
(71)
Applicant(s)
Genentech, Inc.
(72)
Inventor(s)
Camellia W. Adams; Paul J Carter; Brian M Fendly; Austin L. Gurney
(74)
Agent/Attorney
GRIFFITH HACK.GPO BOX 31 25.BRISBANE QLD 4001
(56)
Related Art
EXPERIMENTAL HEMATOLOGY, 24(9),1 996,P260,ABSTRACT NO.1072
OPI DATE 16/03/99
AOJP DATE 13/05/99
APPLN. ID 88312/98
PCT NUMBER PCT/US98/ 17364
INI II II III I Hill
AU9888312
)
(51) International Patent Classification 6 :
C12N 15/13, C07K 16/28, 17/00, A61K
39/395, C07K 19/00, C12N 15/85, 5/10 II
15/62, 15/10
A2
(11) International Publication Number:
(43) International Publication Date:
WO 99/10494
4 March 1999 (04.03.99)
(21) International Application Number: PCT/US98/17364
(22) International Filing Date: 21 August 1998 (21.08.98)
(30) Priority Data:
08/918,148 25 August 1997 (25.08.97) US
(71) Applicant: GENENTECH, INC. [US/US]; 1 DNA Way, South
San Francisco, CA 94080-4990 (US).
(72) Inventors: ADAMS, Camellia, W.; 116C Flynn Avenue,
Mountain View, CA 94043 (US). CARTER, Paul, J.;
1048 Monterey Boulevard, San Francisco, CA 94127 (US).
FENDLY, Brian, M.; 125 Troon Way, Half Moon Bav,
CA 94109 (US). GURNEY, Austin, L.; 1 Debbie Lane,
Belmont, CA 94002 (US).
(74) Agents: SCHWARTZ, Timothy, R. et al.; Genentech, Inc., 1
DNA Way, South San Francisco, CA 94080-4990 (US).
(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE,
GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ,
LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW,
MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ,
TM, TR, TT, UA, UG, UZ, VN, YU, ZW, ARIPO patent
(GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent
(AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent
(AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT,
LU, MC, NL, PT, SE), OAPI patent (BF. BJ. CF, CG, CI.
CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
Published
Without international search report and to be republished
upon receipt of that report.
(54) Title: AGONIST ANTIBODIES TO THE THROMBOPOIETIN RECEPTOR, AND THEIR THERAPEUTIC USES
(57) Abstract
Various forms of c-mpl agonist antibodies are shown to influence the replication, differentiation or maturation of blood cells, especially
megakaryocytes and megakaryocyte progenitor cells. Accordingly, these compounds may be used for treatment of thrombocytopenia.
WO 99/10494
PCT/US98/17364
AGONIST ANTIBODIES TO THE THROMBOPOIETIN RECEPTOR, AND THEIR THERAPEUTIC USES
Field of the Invention
This invention relates to the recombinant synthesis and purification of protein antibodies that influence
5 survival, proliferation, differentiation or maturation of hematopoietic cells, especially platelet progenitor cells
and to antibodies that influence the growth and differentiation of cells expressing a protein kinase receptor.
This invention also relates to the cloning and expression of nucleic acids encoding antibody iigands
(thrombopoietin receptor agonist antibodies) capable of binding to and activating a thrombopoietin receptor
such as c-mpl, a member of the cytokine receptor superfamily. This invention further relates to the use of these
10 antibodies alone or in combination with other cytokines to treat immune or hematopoietic disorders including
thrombocytopenia and to uses in assays.
Background of the Invention
in 1994 several groups reported the isolation and cloning of thrombopoietin (F. de Sauvage et al..
Nature 369:533 (1994): S. Lok et al., Nature 369:565 (1994); TD. Bartley et al., Cell 77:1117 (1994); Y.
15 Sohma et al., FEBS Letters 353:57 (1994); DJ. Kuter et al., Proc. Natl. Acad. Sci. 91:1 1 104 (1994)). This
was the culmination of more than 30 years of research initiated in the late 50' s when Yamamoto (S. Yamamoto.
Acta Haematol Jpn . 20:163-178. (1957)) and Kelemen ( E. Kelemen et al.. Acta Haematol (Basel). 20:350-355
(1958)) proposed that physiological platelet production is controlled by a humoral factor termed
"thrombopoietin"(TPO). Although routinely detected in urine, plasma and serum from thrombocytopenic
20 animals and patients, as well as kidney cell conditioned media, purification of TPO proved to be a daunting task
(for a review see MS. Gordon et al., Blood 80:302 (1992); W. Vainchenker et al., Critical Rev.
Oncology/Hematoiogy 20:165 (1995)). In the absence of purified TPO and the apparent fact that numerous
plieotrophic cytokines affected megakaryocytopoiesis (MS. Gordon et al., Blood 80:302 (1992); W.
Vainchenker et al., Critical Rev. Oncology/Hematoiogy 20:165 (1995)), the existence of a lineage specific
25 factor that regulated platelet production was doubted until the discovery of the orphan cytokine receptor c-Mpl
in 1990 (M. Souyri et al.. Cell 63:1 137 (1990); I. Vigon et al., Proc. Natl. Acad. Sci. 89:5640 (1992)). The
expression of c-Mpl was found to be restricted to progenitor cells, megakaryocytes and platelets, and c-Mpl
antisense oligonucleotides selectively inhibited in vitro megakaryocytopoiesis (M. Methia et al.. Blood 82: 1395
(1993)). From this it was postulated that c-Mpl played a critical role in regulating megakaryocytopoiesis and
30 that its putative ligand may be the long sought TPO (M. Methia et al., supra). Following this discovery several
groups utilizing c-Mpl ligand specific cell proliferation assays and c-Mpl as a purification tool isolated and
cloned the ligand for c-Mpl (F. de Sauvage et al., supra, S. Lok et al, supra; TD. Bartley et al., supra). In
addition two other groups independently reported the purification of the Mpl-ligand using standard
chromatography techniques and megakaryocyte assays (Y. Sohma et al., supra; DJ. Kutere/ al., supra). In the
35 years since its reported discovery numerous studies clearly indicate that the Mpl-ligand possess all the
characteristics that have long been attributed to the purported regulator of megakaryocytopoiesis and
thrombopoiesis and consequently, is now referred to as TPO. The Mpl ligand is currently referred to as either
TPO or as megakaryocyte growth and differentiation factor (MGDF).
-1-
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Human TPO consists of 332 amino acids that can be divided into 2 domains; an amino terminal
domain of 153 amino acids showing 23% identity (50% similarity) to erythropoietin (EPO) and a unique 181
amino acid C-terminal domain that is highly glycosylated ((F. de Sauvage et al., supra; S. Lok et al., supra;
TD. Bartley et al., supra). The EPO-like domain of TPO contains 4 cysteines, 3 of which are conserved with
5 EPO. The first and last and the two middle cysteines form two disulfide bridges, respectively, which are both
required for activity (T. Kato et al., Blood 86 (suppl 1):365 (1995)). None of the Asn-linked glycosylation
sites present in EPO are conserved in the EPO-like domain of TPO, however, the EPO-like domain of
recombinant TPO (rTPO) contains 2-3 O-linked glycosylations (M. Eng et al., Protein Science 5(suppl 1): 105
(1996)). A recombinant truncated form of TPO (rTPO!53), consisting of only the EPO-like domain, is fully
10 functional in vitro, indicating that this domain contains all the required structural elements to bind and activate
Mpl (F. de Sauvage et al., supra; DL. Eaton et al., Blood 84(suppl 1):241 (1994)). The carboxy terminal
domain of TPO contains 6 N-linked and 1 8 O-linked glycosylated sites and is rich in proline, serine and
threonine (M. Eng et al., supra). The function of this domain remains to be elucidated. However, because of
its high degree of glycosylation this region may act to stabilize and increase the half life of circulating TPO.
15 This is supported by the observation that rTPO 153 has a half life of 1 .5 hours compared to 18-24 hours for full
length glycosylated rTPO (GR. Thomas et al., Stem Cells 14(suppl 1) (1996).
The two domains of TPO are separated by a potential dibasic proteolytic cleavage site that is conserved
among the various species examined. Processing at this site could be responsible for releasing the C-terminal
region from the EPO domain in vivo. The physiological relevance of this potential cleavage site is unclear at
20 this time. Whether TPO circulates as an intact full length molecule or as a truncated form is also equivocal.
When aplastic porcine plasma was subjected to gel filtration chromatography, TPO activity present in this
plasma resolved with a Mr. of ~ 1 50,000 ((F. de Sauvage et al., supra). Purified full length rTPO also resolves
at this Mr., whereas the truncated forms resolve with Mr. ranging from 1 8,000-30,000. Using TPO ELISAs that
selectively detect either full length or truncated TPO it has also been shown that full length TPO is the
25 predominant form in the plasma of marrow transplant patients (YG. Meng et al., Blood 86(suppl. 1):313
(1995)).
Prior to the discovery of c-Mpl and the isolation of TPO, it was thought that megakaryocytopoiesis
was regulated at multiple cellular levels (MS. Gordon et al., supra; W. Vainchenker et al., supra; YG. Meng et
al., supra). This hypothesis was based on the observation that certain hematopoietic growth factors stimulated
30 proliferation of megakaryocyte progenitors while others primarily affected maturation (MS. Gordon et al.,
supra; W. Vainchenker et al., supra; YG. Meng et al., supra). Other data indicated that plasma from
thrombocytopenic animals contained distinct activities that either affected proliferation (meg-CSF) or
maturation (TPO) of megakaryocytes (RJ. Hill et al., Exp.Hematol 20:354 (1992)). Wendling and her
colleagues (F. Wendling et al., Nature 369:51 \ (1994)) initially dispelled this theory by demonstrating that all
35 the megakaryocyte colony-stimulating and thrombopoietic activities in thrombocytopenic plasma could be
neutralized by soluble Mpl. This indicated that these activities are due to a single factor, the Mpl-ligand.
Numerous studies have now shown that recombinant forms of TPO not only induce proliferation of progenitor
megakaryocytes but also their maturation ( K. Kaushansky et al., Nature 369:568 (1994); FC. Zeigler et al.,
Blood 84:4045 (1994); VC. Broudy et al.. Blood 85:1719 (1995); JL. Nichol et al., J. Clin. Invest. 95:2973
SUBSTITUTE SHEET (RULE 26)
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(1995) ; N. Banu el al.. Blood 86:133 1 (1995); N. Debili et al.. Blood 86:25 16 (1995); P. Angchaisuksiri et al.,
Br. J. Haematol. 93: 13 (1996); ES. Choi et al., Blood 85:402 (1995)). Human CD34+, CD34+CD41+ cells (FC.
Zeigler et al., supra: VC. Broudy et al., supra; JL. Nichol et al., supra; N. Banu et al., supra;) or purified
murine stem cells (sca+.lin-, kit+) (K. Kaushansky et al., supra; FC. Zeigler et al., supra) cultured with rTPO
5 selectively differentiate to megakaryocytes. rTPO induces the differentiation and proliferation of
megakaryocyte colonies in semisolid cultures and single megakaryocytes in liquid suspension cultures. This
activity appears to be a direct effect of TPO as limiting dilution experiments show a direct correlation between
progenitors seeded and megakaryocytes obtained (N. Debili et al., supra). In addition comparable results are
obtained in serum free or serum containing culture conditions (N. Banu et al., supra; N. Debili et al., supra; P.
10 Angchaisuksiri et al., supra;). These observations indicate that neither accessory cells or serum components
are required for TPO to induce megakaryocyte growth and differentiation in vitro.
The effect of rTPO on the megakaryocyte maturation process is dramatic. rTPO induces highly
purified murine or human progenitor cells in liquid culture to differentiate into very large mature polyploid
megakaryocytes (FC. Zeigler et al., supra; VC. Broudy et al., supra; JL. Nichol et al.. supra: N. Debili ei al..
15 supra). Megakaryocytes from such cultures exhibit ploidy of 4N-16N with ploidy classes of 64N andl28N also
being detected in these cultures (N. Debili et al., supra). In addition, megakaryocytes produced from these
cultures undergo a terminal maturation process and appear to develop proplatelets and shed platelet like
structures into the medium (FC. Zeigler et al., supra; N. Debili et al., supra; ES. Choi el al., supra).
Significantly, the platelets produced from such cultures have been shown to be morphologically and
20 functionally indistinct from plasma-derived platelets (ES. Choi et al., supra).
Although, rTPO appears to act directly on hematopoietic progenitors to induce megakaryocyte
differentiation, it also acts synergistically and additively with early and late acting hematopoietic factors. In
murine megakaryocytopoiesis assays IL-11, kit ligand (KL) or EPO act synergistically and IL-3 and IL-6 act
additively with rTPO to stimulate proliferation of megakaryocyte progenitors (VC. Broudy et al., supra). In
25 human megakaryocytopoiesis assays IL-3 and IL-6 effects are additive to rTPO, while KL acts synergistically
with rTPO (JL. Nichol et al., supra: N. Banu et al., supra; N. Debili et al., supra; P. Angchaisuksiri et al.,
supra). None of the cytokines mentioned above affect the megakaryocyte maturational activity of rTPO.
The initial studies with rTPO clearly indicate that TPO predominantly affects the megakaryocyte
lineage. However, like all other hematopoietic regulators, TPO affects other hematopoietic lineages as well. In
30 the presence of EPO, rTPO has been shown to enhance erythroid burst (BFU-E) formation in human CD34+
colony assays ( M. Kobayashi et al., Blood 86:2494 (1995); T. Papayannopoulou et al., Blood 87:1833
(1996) ). The burst promoting activity of rTPO is comparable to GM-CSF and KL and increases both the
number and size of BFU-E colonies (M. Kobayashi et al., supra). In addition rTPO also stimulates CFU-E
development, indicating that TPO acts on both early and late erythroid progenitors ( M. Kobayashi et al., supra;
35 T. Papayannopoulou et al., supra ). In the absence of EPO, however, rTPO has no effect on erythropoiesis.
An effect of rTPO on myeloid colony growth in normal hematopoietic cultures has not been demonstrated in
vitro, however.
rTPO has a dramatic effect on platelet production when administered to normal animals.
Pharmacological doses of recombinant forms of TPO cause as much as a 10 fold increase in platelet levels in
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mice and non-human primates (EF. Winton et al.. Exp. Hematol. 23:879 (1995); AM. Farese et al.. Blood 86:54
(1995); KH. Sprugel et al.. Blood 86(suppl 1):20 (1995); LA. Harker et al., Blood 87:1833 (1996); K.
Kaushansky et al., Exp. Hematol. 24:265 (1996); TR. Ulich et al., Blood 87:5006 (1996); K. Ault et al., Blood
86(suppl 1): 367 (1995); NC. Daw et al., Blood 86 (suppl 1):5006 (1995)). This effect of rTPO is due to an
5 increase in the synthesis of new platelets as reticulated platelets increase within 24 hours after rTPO
administration (K. Ault et al., supra). Preceding this effect is a dramatic increase in CFU-MK in both the
marrow and spleen ( AM. Farese et al., supra; K. Kaushansky et al., supra; TR. Ulich et al., supra).
Megakaryocytes from rTPO treated animals exhibit a higher mean ploidy and are larger in size than
megakaryocytes from control animals. These later two observations again demonstrate the proliferative and
10 maturational activities of TPO on the megakaryocytic lineage. Because the effect of TPO on megakaryocytes
precedes its effect on platelet production it has been suggested that TPO primarily affects megakaryocyte
progenitors rather than inducing platelet release from mature megakaryocytes (NC. Daw et al., supra). No
significant effect on red blood cell (RBC) or white blood cell (WBC) production occurs in normal animals
following rTPO administration. However, rTPO treatment caused an expansion of BFU-E and CFU-GM and a
15 redistribution CFU-E in normal mice (K. Kaushansky et al., supra) and expanded CFU-mixed in rhesus
monkeys (AM. Farese et al., supra).
Even though rTPO dramatically stimulates platelet production, it only has a modest effect on platelet
function. In vitro studies show that rTPO has no effect on platelet aggregation itself, but does enhance agonist
induced aggregation (G. Montrucchio et al., Blood 87:2762 (1996); A. Oda et al.. Blood 87:4664 (1996); CF.
20 Toombs et al., Thromb. Res. 80:23 (1995); CF. Toombs et al., Blood 86(suppl 1):369 (1995)). rTPO appears
to sensitize platelets making them moderately more responsive to aggregation agonist. This raises the
possibility that rTPO may have prothrombotic effects in vivo. However, an increase in thrombotic episodes in
animals treated with rTPO has never been observed, even when platelet levels were 4-10 fold above normal. In
vivo thrombosis models also indicate that elevated platelet levels following rTPO treatment is not associated
25 with an increase in platelet dependent thrombosis (LA. Harker et al., supra; CF. Toombs et al., supra). These
results indicate that stimulation of platelet production by rTPO will unlikely be associated with an increase in
thrombo-occulsive events.
The involvement of c-MpI and TPO in the control of platelet production and its effect on other
hematopoietic lineages is further demonstrated by the phenotype of mice deficient in either the c-mpl or the
30 TPO genes (WS. Alexander et al.. Blood 87:2162 (1996); FJ. de Sauvage et al., J.Exp.Med. 183:651 (1996);
AL. Gumey et al., Science 265:1445 (1994)). In both cases a dramatic 85 to 90% drop in platelet counts is
observed with a similar decrease of megakaryocytes in the spleen and bone marrow. In addition, the
megakaryocytes of the knockout mice are smaller and exhibit a lower ploidy than those of control mice. The
similarity in phenotype observed for these knock-outs (KO) indicates that the system is non-redundant and that
35 there is probably only one receptor for TPO and one ligand for c-Mpl. Although the platelet number is reduced
in the KO mice their platelets appear normal, both structurally and functionally, and are sufficient to prevent
overt bleeding. The genes and factors involved in the production of this basal level of platelets and
megakaryocytes still remain to be identified. However, treatment of either the TPO or c-mpl knockout mice
with other cytokines with megakaryopoietic activity (1L-6.IL-11 and stem cell factor) results in a modest
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stimulation of platelet production (AL. Gurney et ah, supra). This suggest that these cytokines do not require
TPO or c-mpl to exert their thrombopoietic activity and, therefore, may be involved in the maintenance of a
basal level of megakaryocytes and platelets.
Comparison of CFU-megakaryocyte (CFU-Meg) from TPO or c-mpl deficient and normal mice shows
5 that the number of megakaryocytes progenitors is decreased in both knock-outs compared to control, suggesting
that TPO acts on very early megakaryocyte progenitors. In addition, both erythroid and myeloid progenitors
are also reduced in the TPO and c-Mpl knockout mice (WS. Alexander et al., supra; K. Carver- Moore et al.,
88:803 (1996)). This reduction in progenitors from all lineages indicates that TPO probably acts on a very early
pluripotent progenitor cell. The involvement of TPO and c-Mpl at an early stage of hematopoiesis correlates
10 with the detection of c-Mpl expression in AA4+ Sca+ murine stem cell population (FC. Zeigler et al., supra).
The effect of TPO on this most primitive stem cell population still remains to be investigated, however,
preliminary data indicate that TPO may directly affect the proliferation of primitive murine hematopoietic stem
or progenitor cells (E. Stinicka et al., Blood 87:4998 (1996); M. Kobayashi et al., Blood 88:429 (1996); H. Ku
et al., Blood 87:4544 (1996)). This, in part, may explain the effect TPO has on erythropoiesis and myelopoiesis
1 5 in vitro and in vivo.
It has long been observed that an inverse correlation exists between plasma megakaryopoietic and
thrombopoietic activity and platelet levels (reviewed in TP. McDonald, Am. J. Pediatr. Hematol./Oncol. 14:8
(1992)). TPO specific ELISAs and cell proliferation assays have now confirmed that TPO levels increase and
decrease inversely with platelet mass (JL. Nichol et al., supra; EVB. Emmons et al., Blood 87:4068 (1996); H.
20 Oh et al., Blood 87:4918 (1996); M. Chang et al.. Blood 86(suppl 1):368 (1995)). Unlike erythropoietin,
however, TPO does not appear to be regulated at the transcriptional level, but rather by platelet mass. This was
initially proposed de Gabriele and Pennington (G. de Gabriele et al., Br. J. Haematol. 13:202 (1967); G. de
Gabriele et al., Br. J. Haematol. 13:210 (1967)) and subsequently confirmed by Kuter and Rosenberg (DJ.
Kuter et al.. Blood 84:1464 (1994)) who showed direct regulation of circulating TPO levels by exogenously
25 administering platelets to thrombocytopenic mice. More recently, it was demonstrated that TPO mRNA levels
in thrombocytopenic mice are not increased even though TPO levels are elevated by at least 10 fold (PJ.
Fielder et al., Blood 87:2154 (1996); R. Stoffel et al.. Blood 87:567 (1996)). In addition, the gene dosage
effect observed in TPO heterozygous knockout mice refute the regulation of TPO production by platelet mass
(FJ. de Sauvage et al., supra). Taken together, these results strongly support the hypothesis that TPO expression
30 is constitutive and it is the sequestering by platelets that regulates TPO levels. Platelets bind TPO with high
affinity (Kd(100-400pM) and internalize and degrade TPO (PJ. Fielder et al., supra). Platelets from c-Mpl
knockout mice do not bind TPO and the clearance of TPO by these mice is 5 fold slower than that observed for
wild type mice (PJ. Fielder et al., supra). These results indicate that TPO clearance is mediated by platelet
binding via c-Mpl. It is also likely that megakaryocyte mass plays a role in regulating circulating TPO levels.
35 This is supported by the observation that both ITP patients and mice deficient in the NF-E2 transcription factor
are highly thrombocytopenic, exhibit megakaryocytosis, but have normal TPO levels (EVB. Emmons et al.,
supra; RA. Shivdasani et al., Cell 81:695 (1995)). In situ studies with radiolabeled TPO show that marrow
megakaryocytes of the NF-E2 mice bind significant amounts of labeled TPO (RA. Shivdasani et al., Blood
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submitted (1996)). The phenotype of the ITP and NF-E2 knockout mice, therefore, suggest that binding of
TPO to megakaryocytes may also regulate TPO levels.
The dramatic effect of rTPO on platelet production in normal mice and monkeys and subsequent
clinical trials indicate that rTPO is clinically useful in alleviating thrombocytopenia associated with
5 myelosuppressive and myeloablative therapies for cancer patients. In several myelosuppressive and
myeioablative murine and monkey preclinical models recombinant forms of TPO have been shown to
significantly affect platelet recovery. In mice treated with carboplatin and sublethal irradiation in combination
(JP Leonard et al., Blood 83:1499 (1994)), daily treatment with rTPO both reduced the severity of the platelet
nadir and accelerated platelet recovery by 10-12 days when compared to excipient treated animals (GR. Thomas
10 et al., supra; K. Kaushansky et al., supra; MM. Hokom et al., Blood 86:4486 (1995)). Similar results were
obtained in a murine sublethal irradiation model (GR. Thomas et al., supra). In murine myeloablative
transplantation models rTPO has been shown to reduce the extent of the nadir and accelerate platelet recovery
by 2-3 weeks (GR. Thomas et al., supra; K. Kabaya et al., Blood 86(suppl 1): 1 14 (1995); G. Molineux et al..
Blood 86(suppl 1):227 (1995)). Treatment of sublethally irradiated rhesus monkeys with rTPO accelerated
15 piatelet recovery by 3 weeks and prevented platelet nadirs below 40,000 (AM. Farese et al., J. Clin. Invest.
97:2145 (1996); KJ. Neelis et at., Blood 86(suppl 1):256 (1995)). Even more impressively, rTPO completely
prevented post-chemotherapy thrombocytopenia following the treatment of rhesus monkeys with hepsulfam
(AM. Farese et al., supra). In contrast to these promising results, two groups have reported that rTPO had no
effect on the hematopoietic recovery of lethally irradiated mice or monkeys rescued with a marrow transplant
20 (KJ. Neelis et al., supra; WE. Fibbe et al.. Blood 86:3308 (1995)). The reason for this discrepancy is unclear,
however it is possible that lethal radiation may destroy stromal cells or components essential for TPO activity in
vivo. In support of this, lethally irradiated mice transplanted with marrow cells from rTPO treated donor mice
show accelerated recovery of platelets and RBCs, however, post-transplant administration of rTPO had no
further effect on this accelerated recovery (WE. Fibbe et al., supra). This result suggests that although the
25 transplanted cell population was enriched for megakaryocyte progenitors, TPO had no effect on these
progenitors in a lethally irradiated marrow.
Although rTPO only modestly affects erythroid and myeloid lineages in normal mice it dramatically
accelerates the recovery of all progenitor classes in myeiosuppressed mice and monkeys resulting in a
significant acceleration of RBC and WBC recovery (K. Kaushansky et al., supra; AM. Farese et al., supra; K.
30 Kaushansky et al., J. Clin. Invest. 96:1683 (1995)). The effect of rTPO on neutrophil recovery has been shown
to be additive to that of G-CSF (AM. Farese et al., supra). These results indicate that the clinical utility of
rTPO may be broader than originally anticipated.
The difference between the effect of rTPO on hematopoiesis in normal and myeiosuppressed animals
is likely due to the change in the cytokine environment that occurs following myelosuppressive therapy. It is
35 likely that elevated levels of EPO, G-CSF or other cytokines essential for erythropoiesis and myelopoiesis
present following myelosuppressive treatment interact with rTPO to have a multilineage effect (K. Kaushansky
et al., supra). In normal mice the level of these cytokines are insufficient and the effects of rTPO on erythroid
and myeloid lineages are less significant. This hypothesis is supported by the above mentioned synergistic
interaction of rTPO and EPO to stimulate in vitro erythropoiesis (ES. Choi et al., supra). It has also been
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proposed that production of hemopoietic factors from megakaryocytes themselves may also play a role in the
multilineage effect of rTPO (AM. Farese et al., supra).
In the above mentioned animal studies rTPO was administered daily for 14-28 days, which was based
on previous experience in dosing other hematopoietic growth factors. However, it has recently been shown that
5 a single dose of rTPO following myelosuppressive treatment of mice with carboplatin and sublethal irradiation
is as effective as multiple doses in reducing nadirs and accelerating platelet and RBC recovery (GR. Thomas et
al, supra). This effect is likely due to the potency and long half life of rTPO
(GR. Thomas et al, supra). This is supported by the fact that single doses of unglycosylated rTPO 153
are not effective in this model. These observations indicate that the frequency of rTPO dosing required to affect
10 hematopoietic recovery following myelosuppressive treatment may be significantly less than that for other
currently used cytokines
Early results from human clinical trails show that rTPO also stimulates platelet production in humans.
In phase I trials, a pegylated and truncated form of rTPO (MGDF) administered daily for 10 days at 0.03 - 5.0
Hg/kg to cancer patients prior to chemotherapy caused up to a four fold increase in circulating platelet levels (R.
15 Basser et al., Blood 86(suppl 1): 257 (1995); JEJ. Rasko et al.. Blood 86(suppl I):497 (1995)). Similarly,
patients given a single dose of rTPO had platelet levels increase by four fold (S. Vaden-Raj et al.. Stimulation
of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in cancer
patients. Submitted. (1996)). In both studies platelet increases are observed by day four and peak about 12-16
days later. No drug related toxicity's were reported and, although platelet levels greater then lX106/fil were
20 observed in some of the patients, no thrombotic events were observed. This indicates that TPO will be well
tolerated in humans. In myelosuppressed patients, pegylated rTPO 153 (MGDF) given post chemotherapy has
been shown to reduce the extent of the platelet nadir following chemotherapy (G. Begley et al., Proceedings of
ASCO 15:271 (1996); M. Fanucchi et al., Proceedings of ASCO 15:271 (1996)). As seen in the preclinical
animals studies, TPO also expanded marrow progenitors of megakaryocyte, erythroid, myeloid and
25 multipotential lineages (S. Vaden-Raj et al.. supra). This later observation suggests that rTPO may be useful as
a priming agent.
It is believed that the proliferation and maturation of hematopoietic cells is tightly regulated by factors
that positively or negatively modulate pluripotential stem cell proliferation and multilineage differentiation.
These effects are mediated through the high-affinity binding of extracellular protein factors (ligands) to specific
30 cell surface receptors. These cell surface receptors share considerable homology and are generally classified as
members of the cytokine receptor superfamily. Members of the superfamiiy include receptors for: IL-2 (b and
g chains) (Hatakeyama et al., Science, 244:551-556 (1989); Takeshita et al., Science, 257:379-382 (1991)), IL-
3 (Itoh et al., Science, 247:324-328 (1990); Gorman et al., Proc. Natl. Acad. Sci. USA, 87:5459-5463 (1990);
Kitamura et al.. Cell, 66:1165-1174 (1991a); Kitamura et al.. Proc. Natl. Acad. Sci. USA, 88:5082-5086
35 (1991b)), IL-4 (Mosley et al.. Cell, 59:335-348 (1989), IL-5 (Takaki et al., EM BO J., 9:4367-4374 (1990);
Tavemier et al. Cell, 66:1 175-1184 (1991)), IL-6 (Yamasaki et al, Science, 241:825-828 (1988); Hibi et al,
Cell, 63:1 149-1157 (1990)), IL-7 (Goodwin et al.. Cell, 60:941-951 (1990)), IL-9 (Renault et al., Proc. Natl.
Acad. Sci. USA, 89:5690-5694 (1992)), granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gearing
et al, EMBO J.. 8:3667-3676 (1991); Hayashida et al. Proc. Natl. Acad. Sci. USA, 244:9655-9659 (1990)),
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granulocyte colony-stimulating factor (G-CSF) (Fukunaga et al, Cell, 61:341-350 (1990a); Fukunaga et al,
Proc. Natl. Acad. Sci. USA, 87:8702-8706 (1990b); Larsen et al., J. Exp. Med, 172:1559-1570 (1990)), EPO
(D' Andrea et al.. Cell, 57:277-285 (1989); Jones et al.. Blood, 76:31-35 (1990)), Leukemia inhibitory factor
(L1F) (Gearing et al., EM BO J., 10:2839-2848 (1991)), oncostatin M (OSM) (Rose et al., Proc. Natl. Acad. Sci.
5 USA, 88:864 1 -8645 ( 1 99 1 )) and also receptors for prolactin (Boutin et al., Proc. Natl. Acad. Sci. USA, 88:7744-
7748 (1988); Edery et al., Proc. Natl. Acad. Sci. USA, 86:21 12-21 16 (1989)), growth hormone (GH) (Leung et
al. Nature, 330:537-543 (1987)) and ciliary neurotrophic factor (CNTF) (Davis et al.. Science, 253:59-63
(1991).
Members of the cytokine receptor superfamily may be grouped into three functional categories (for
10 review see Nicola et al., Cell, 67:1-4 (1991)). The first class comprises single chain receptors, such as
erythropoietin receptor (EPO-R) or granulocyte colony stimulating factor receptor (G-CSF-R), which bind
ligand with high affinity via the extracellular domain and also generate an intracellular signal. A second class
of receptors, so called a-subunits, includes interleukin-6 receptor (IL6-R), granulocyte-macrophage colony
stimulating factor receptor (GM-CSF-R), interleukin-3 receptor (IL3-Ra) and other members of the cytokine
15 receptor superfamily. These a-subunits bind ligand with low affinity but cannot transduce an intracellular
signal. A high affinity receptor capable of signaling is generated by a heterodimer between an a-subunit and a
member of a third class of cytokine receptors, termed b-subunits, e.g., b c , the common b-subunit for the three a-
subunitsof IL-3-R, IL-5-R and GM-CSF-R (Nicola N. A. et. al. Cell 67:1-4 (1991))
Evidence that mpl is a member of the cytokine receptor superfamily comes from sequence homology
20 (Gearing, EMBO J., 8:3667-3676 (1988); Bazan, Proc. Natl. Acad. Sci. USA, 87:6834-6938 (1990); Davis et
a!.. Science, 253:59-63 (1991) and Vigon et al., Proc. Natl. Acad. Sci. USA, 89:5640-5644 (1992)) and its
ability to transduce proliferative signals.
Deduced protein sequence from molecular cloning of murine c-mpl reveals this protein is homologous
to other cytokine receptors. The extracellular domain contains 465 amino acid residues and is composed of two
25 subdomains each with four highly conserved cysteines and a particular motif in the N-terminal subdomain and
in the C-terminal subdomain. The iigand-binding extracellular domains are predicted to have similar double b-
barrel fold structural geometries. This duplicated extracellular domain is highly homologous to the signal
transducing chain common to IL-3, IL-5 and GM-CSF receptors as well as the low-affinity binding domain of
LIF (Vigon et al., Oncogene, 8:2607-2615 (1993)). Thus mpl may belong to the low affinity ligand binding
30 class of cytokine receptors.
A comparison of murine mpl and mature human mpl P, reveals these two proteins show 81% sequence
identity. More specifically, the N-terminus and C-terminus extracellular subdomains share 75% and 80%
sequence identity respectively. The most conserved mpl region is the cytoplasmic domain showing 91% amino
acid identity, with a sequence of 37 residues near the transmembrane domain being identical in both species.
35 Accordingly, mpl is reported to be one of the most conserved members of the cytokine receptor superfamily
(Vigon supra).
Activation of certain hematopoietic receptors is believed to cause one or more effects including;
stimulation of proliferation, stimulation of differentiation, stimulation of growth and inhibition of apoptosis
(Libol et al Proc. Natl. Acad. Sci. 248:378 (1993). Activation of hematopoietic receptors upon ligand binding
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may be due 10 dimerization of two or more cop.Ses.Qf the receptor. In addition to the naturally occurring ligand
causing this dimeriration, agonist antibodies may also activate receptors by crosslinking or otherwise causing
dimcriwtion of a receptor. Such antibodies are useful for the same indications as the natural ligand and may
have advantageous properties such as a longer half-life. An example of a monoclonal antibody to a cytokine
5 receptor that activates the erythropoietin receptor (£PO-R) is described in WO 96/03438 (published 8 February
1996). These agonist antibodies in EPO-R arc about 3-4 orders of magnitude weaker in activity based on
weight than the natural EPO ligand.
There is a current and continuing need to isolate and identify molecules, especially antibodies,
fragments and derivatives thereof, capable of stimulating proliferation, differentiation and maturation and/or
10 modulation of apoptosis or cells, for example hematopoietic cells, including megakaryocytes or their
predecessors for therapeutic use in the treatment of hematopoietic disorders including thrombocytopenia.
Summary Of The Invention
According to a first aspect of the present invention there is provided an agonist antibody or fragment thereof
which binds to a thrombopoietin receptor, which is selected from the group consisting of Abi, Ab2, Ab3, Ab4, AbS and
1 5 Ab6, wherein each Abl-AbS comprises a VH and VL chain, each VH and VL chain comprising CDft amino acid
sequences designated CDR1 , CDR2 and CDR3 separated by framework amino acid sequences, the amino acid
sequences of each CDR in each VH and VL chain of Ab1-Ab6 selected according to the following table:
20 • - .'
30
35
i
I
05^11/2002 15:31 GRIFFITH HACK ■> IP AUSTRALIA PT
NO. 601 D004
Abl:
DNA
protein
DNA
protein
Ab2:
DNA
protein
DNA
protein
Ab3:
DNA
protein
DNA
protein
AM:
DNA
protein
DNA
protein
AbS:
DNA
protein
vhCdri
(seq id no: i)
(SEQ ID NO: 2)
VLCDRI
(SEQ ID NO: 7)
(SEQ ID NO: 8)
VHCDRI
(SEQ ID NO: 13)
(SEQ ID NO: 14)
ViCDRl
(SEQ ID NO: 19)
(SEQ ID NO: 20)
VH CDRI
(SEQ ID NO: 25)
(SEQ ID NO: 26)
VlCDRI
(SEQ ID NO: 19)
(SEQ ID NO: 20)
VH CDRI
(SEQ ID NO: 25)
(SEQ ID NO: 26)
VtCD R l
(SEQ ID NO: 35)
(SEQ ID NO: 20)
VHCDRI
(SEQ ID NO: 36)
(SEQ ID NO: 37)
Table 1
VH CDR2
(SEQ ID NO: 3)
(SEQ ID NO: 4)
VLCDR2
(SEQ ID NO; 9)
(SEQ ID NO: 10)
YH CDR2
(SEQ ID NO: IS)
(SEQ ID NO: 16)
(SEQ ID NO: 21)
(SEQ ID NO: 22)
VHCDR2
(SEQ ID NO: 27)
(SEQ ID NO: 2S)
YL.CDR2 .
(SEQ ID NO: 21)
(SEQ ID NO: 22)
VH CDR2
(SEQ ID NO: 31)
(SEQ ID NO: 32)
VL CDR2
(SEQ ID NO: 21)
(SEQ ID NO: 22)
VH CDR2
(SEQ ID NO: 38)
(SEQ ID NO: 39)
VH CDR3
(SEQ ID NO: 5)
(SEQ ID NO: 6)
V ]_CDR3
(SEQ ID NO: 11)
(SEQ ID NO: 12)
VH CDR 3
(SEQ ID NO: 17)
(SEQ ID NO: 18)
VLCDR3
(SEQ ID NO: 23)
(SEQ ID NO: 24)
VH CDRJ
(SEQ ID NO: 29)
(SEQ ID NO: 30)
VLCDR3
(SEQ ID NO: 23)
(SEQ ID NO: 24)
VH CDR3
(SEQ ID NO: 33)
(SEQ ID NO: 34)
VLCDR3
(SEQ ID NO; 23)
(SEQ ID NO: 24)
VH CDR3
(SEQ ID NO: 40)
(SEQ ID NO: 41)
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05^11/2002 15:31 GRIFFITH HACK ■» IP AUSTRALIA PT
NO. 601 0005
VL CDR1
VL.CDR2
VLCDR3
DNA
(SEQ ID NO: 19)
(SEQ ID NO: 2t)
(SEQ ID No: 23)
protein
(SEQ ID NO: 20)
(SEQ ID NO: 22)
(SEQ ID No. Zh)
AW:
VH CDR1
VH C0R2
VHCDR3
DNA
(SEQ ID NO: 42)
(SEQ ID NO: 44)
(SEQ ID No: 46)
protein
(SEQ ID NO: O)
(SEQ ID NO: 45)
(SEQ ID No: 47)
vlCdri
VLCDR2
VLCDR3
DNA
(SEQ ID NO: 48)
(SEQ ID NO: SO)
(SEQ 10 No: 52)
protein
(SEQ ID NO: 49)
(SEQ ID NO: 51)
(SEQ ID No: 53)
According to a second aspect of the present Invention there is provided an agonist antibody or fragment
thereof which binds to a thrombopoietin receptor, wherein said antibody or fragment thereof comprises an amino acid
sequence which is selected from the group consisting of 12E1 0, 12B5, 10F6 and 12D5 as set forth in Fig. 1 .
Preferably, the thrombopoietin receptor is mammalian c-mpl. more preferably, human c-mpl.
Usually the antibody will be a full length antibody such K an tgG antibody. Suitable
representative fragment agonist antibodies include Fv, ScFv, Fab. F(aV), fragments, as welt as diabodics and
linear antibodies. These fragments may be fused to other sequences including, for example, the F~ or Fc region
of an antibody, a "leucine zipper" or other sequences including pegylatcd sequences or Fc mutants used to
improve or modulate half-life. Normally the antibody is a human antibody and may be a non-naturslly
occurring antibody, including affinity matured antibodies. Representative antibodies that activate c-mpl are
selected from the group 12EI0, 12B5, IOF6 and 12DS, and affinity matured derivatives thereof. Other
preferred agonist antibodies to c-mpl are selected from the group consisting of Abl, Ab2. Ab3. Ab4, Ab5 and
Ab6, wherein each Abl -AM contains a VH and VL chain and each VH and VL chain contains
complementarity determining region (CDR) amino acid sequences designated CDRl, CDR2 and GDR3
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GRIFFITH HACK
121009
10
Other preferred c-mpl agonist antibodies of this invention include those that activate platelets in a
manner similar to TPO or in a manner similar to ADP, collagen and the like. Optionally the c-mpl agonist
antibodies of this invention do not activate platelets. The c-mpl agonist antibodies of this invention are used in a
manner similar to TPO.
In another embodiment, substantially pure single chain antibodies arc provided which bind to and act
as agonist or antagonist antibodies to a cytokine receptor or to a kinase receptor.
The invention also provides a method of obtaining these antibodies, in particular a method of screening
a library of phage displayed antibodies, preferably human single chain antibodies.
The invention also provides compositions containing antibodies of the invention together with a
pharmaceutical^ acceptable carrier and methods ot stimulating proliferation, differentiation or growth of
megakaryocytes and/or Increasing platelet production by contacting megakaryocytes with antibodies of the invention.
Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in a
non-exclusive sense, except where the context requires otherwise.
It will be clearly understood that, although a number of prior art publications are referred to herein, this
reference does not constitute an admission that any of these documents forms part of the common general Knowledge
15 in the art, in Australia or in any other country.
Brief Description Of The Drawings
Fig. I shows examples of single chain antibody (scFv) fragments denominated I0F6, SES, 10D10,
12BS, I2DS and I2EI0 having sequences for CDRs and framework regions.
Fig. 2 illustrates a method for the construction of a phage library containing single-chain antibodies
fused to a coat protein of a phage. •
Fig- 3 shows a single-chain antibody displayed as. a fusion protein on coat protein 3 of a filamentous
phage. • ' ■'
Fig. 4 illustrates a method of selecting scFv in a phage library by one or more binding selection cycles.
Fig. 5 illustrates a method or panning high affinity phage using biotinylatcd antigen and streptavidin
coated paramagnetic beads.
Fig. 6 shows a process for identifying c-mpl binding phage using a phage EL ISA method.
Fig. 7 illustrates DNA fingerprinting of clones to determine diversity by BstNl restriction enzyme
analysis.
Fig. 8A-C show a typical BstNl analysis on a 3 % agarose gel; see Example 2.
Fig. 9 shows the results of agonist antibodies relative to TPO in the KIRA-ELISA assay.
Fig. 10A-F show the results of TPO-anlibody competitive binding assays for HU-03 cells. See
Example 1.
20
25
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Fig. 1 1 shows activity for MuSK agonist antibodies of Example 5.
Detailed Description Of The Preferred Embodiments
L Definitions
In general, the following words or phrases have the indicated definition when used in the description,
S examples, and claims.
The terms "agonist" and "agonistic" when used herein refer to or describe a molecule which is capable
of, directly or indirectly, substantially inducing, promoting or enhancing cytokine biological activity or
cytokine receptor activation.
"Agonist antibodies"(aAb) are antibodies or fragments thereof that possess the property of binding to
10 a cytokine superfamily receptor and causing the receptor to transduce a survival, proliferation, maturation
and/or differentiation signal. Included within the definition of transducing a survival signal is a signal which
modulates cell survival or death by apoptosis. To be therapeutically useful the agonist antibodies of this
invention will be capable of inducing or causing survival, proliferation, maturation or differentiation at a
concentration equal to or not less than 2 orders of magnitude (100-fold) below that of the natural in vivo ligand
15 on a weight basis.
"Activate a receptor", as used herein, is used interchangeably with transduce a growth, survival,
proliferation, maturation and/or differentiation signal.
"Activate platelets", as used herein, means to stimulate platelets to make them more likely to aggregate
by comparison to unactivated platelets. For example, ADP and collagen are substances known to activate
20 platelets.
"Affinity matured antibodies" are antibodies that have had their binding affinity and/or biological
activity increased by altering the type or location of one or more residues in the variable region. An example of
alteration is a mutation which may be in either a CDR or a framework region. An affinity matured antibody
will typically have its binding affinity increased above that of the isolated or natural antibody or fragment
25 thereof by from 2 to 500 fold. Preferred affinity matured antibodies will have nanomolar or even picomolar
affinities to the receptor antigen. Affinity matured antibodies are produced by procedures known in the art.
Marks, J. D. et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain
shuffling. Random mutagenesis of CDR and/or framework residues is described by; Barbas, C. F. et al. Proc
Nat. Acad. Sci. USA 91:3809-3813 (1994), Schier, R. et al. Gene 169:147-155 (1995), Yelton, D. E. el al. J.
30 Immunol. 155:1994-2004 (1995), Jackson, J.R. el al. J. Immunol. 154(7):33 10-9 (1995), and Hawkins, R.E. et
al, J. Mot. Biol. 226:889-896 (1992).
"Cytokine" is a generic term for proteins released by one cell population which act on another cell as
intercellular mediators. Examples of such cytokines are lymphokines. monokines, and traditional polypeptide
hormones. Included among the cytokines are growth hormone, insulin-like growth factors, human growth
35 hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine,
insulin, proinsulin, reiaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH),
thyroid stimulating hormone (TSH), and leutinizing hormone (LH), hematopoietic growth factor, hepatic
growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-a (TNF-a and TNF-
b) mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial
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f
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growth factor, integrin. nerve growth factors such as NGF-b, platelet-growth factor, transforming growth
factors (TGFs) such as TGF-a and TGF-b, insulin-like growth factor-I and -II, erythropoietin (EPO),
osteoinductive factors, interferons such as interferon-a, -b, and -g, colony stimulating factors (CSFs) such as
macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granuIocyte-CSF (G-CSF),
5 thrombopoietin (TPO). interleukins (IL's) such as IL- 1, IL-la, IL-2, IL-3, IL-4, 1L-5, IL-6, IL-7, IL-8, IL-9, IL-
1 1, IL-12 and other polypeptide factors including LIF, SCF, and kit-Iigand. As used herein the foregoing terms
are meant to include proteins from natural sources or from recombinant cell culture. Similarly, the terms are
intended to include biologically active equivalents; e.g., differing in amino acid sequence by one or more amino
acids or in type or extent of glycosylation.
10 "Cytokine superfamily receptors" and "hematopoietic growth factor superfamily receptors" are used
interchangeably herein and are a group of closely related glycoprotein cell surface receptors that share
considerable homology including frequently a WSXWS domain and are generally classified as members of the
cytokine receptor superfamily (see e.g. Nicola et al, Cell, 67:1-4 (1991) and Skoda, R.C. et al EMBO J.
12:2645-2653 (1993)). Generally, these receptors are interleukins (IL) or colony-stimulating factors (CSF).
15 Members of the superfamily include, but are not limited to, receptors for: IL-2 (b and g chains) (Hatakeyama et
al., Science, 244:551-556 (1989); Takeshita et al.. Science, 257:379-382 (1991)), IL-3 (Itoh et al., Science,
247:324-328 (1990); Gorman et al., Proc. Natl. Acad. Sci. USA, 87:5459-5463 (1990); Kitamura et al.. Cell,
66:1 165-1 174 (1991a); Kitamura et al., Proc. Natl. Acad. Sci. USA. 88:5082-5086 (1991b)), IL-4 (Mosley et
al.. Cell, 59:335-348 (1989), IL-5 (Takaki et al.. EMBO J., 9:4367-4374 (1990); Tavemier et al.. Cell, 66: 1 1 75-
20 1 184 (1991)), IL-6 (Yamasaki et al.. Science, 241:825-828 (1988); Hibi et al.. Cell, 63:1 149-1 157 (1990)), IL-7
(Goodwin et al.. Cell, 60:941-951 (1990)), IL-9 (Renault et al. Proc. Natl. Acad. Sci. USA, 89:5690-5694
(1992)), granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gearing et al.. EMBO J., 8:3667-3676
(1991); Hayashida et al.. Proc. Natl. Acad. Sci. USA, 244:9655-9659 (1990)), granulocyte colony-stimulating
factor (G-CSF) (Fukunaga et al. Cell, 61:341-350 (1990a); Fukunaga et a!., Proc. Natl. Acad. Sci. USA.
25 87:8702-8706 (1990b): Larsen et al.. J. Exp. Med., 172:1559-1570 (1990)), EPO (D'Andrea et al. Cell,
57:277-285 (1989); Jones et al.. Blood, 76:31-35 (1990)), Leukemia inhibitory factor (LIF) (Gearing et al..
EMBO J., 10:2839-2848 (1991)), oncostatin M (OSM) (Rose et al. Proc. Natl. Acad. Sci. USA, 88:8641-8645
(1991)) and also receptors for prolactin (Boutin et al, Proc. Natl. Acad. Sci. USA, 88:7744-7748 (1988); Edery
et al, Proc. Natl Acad. Sci USA, 86:2112-2116 (1989)), growth hormone (GH) (Leung et al. Nature.
30 330:537-543 (1987)), ciliary neurotrophic factor (CNTF) (Davis et al. Science, 253:59-63 (1991) and c-MpI
(M. Souyri et al, Cell 63:1 137 (1990); I. Vigon et al, Proc. Natl. Acad. Sci. 89:5640 (1992)).
"Thrombocytopenia" in humans is defined as a platelet count below 150 X 10^ per liter of blood.
"Thrombopoietic activity" is defined as biological activity that consists of accelerating the
proliferation, differentiation and/or maturation of megakaryocytes or megakaryocyte precursors into the platelet
35 producing form of these cells. This activity may be measured in various assays including an in vivo mouse
platelet rebound synthesis assay, induction of platelet cell surface antigen assay as measured by an anti-platelet
immunoassay (anti-GPIIbHI a ) f° r a human leukemia megakaryoblastic cell line (CMK), and induction of
polyploidization in a megakaryoblastic cell line (DAMI). A "thrombopoietin receptor" is a mammalian
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polypeptide receptor which, when activated by a ligand binding thereto, includes, causes or otherwise gives rise
to "thrombopoietic activity" in a cell or mammal, including a human.
"Control sequences" when referring to expression means DNA sequences necessary for the expression
of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for
5 prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and
possibly, other as yet poorly understood sequences. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.
"Operably linked" when referring to nucleic acids means that the nucleic acids are placed in a
functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory
10 leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is
positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being
linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However,
15 enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If
such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accord with conventional
practice.
"Exogenous" when referring to an element means a nucleic acid sequence that is foreign to the cell,
or homologous to the cell but in a position within the host cell nucleic acid in which the element is ordinarily
20 not found.
"Cell," "cell line," and "cell culture" are used interchangeably herein and such designations include all
progeny of a cell or cell line. Thus, for example, terms like "transformants" and "transformed cells" include the
primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent
25 mutations. Mutant progeny that have the same function or biological activity as screened for in the originally
transformed cell are included. Where distinct designations are intended, it will be clear from the context.
"Plasmids" are autonomously replicating circular DNA molecules possessing independent origins of
replication and are designated herein by a lower case "p" preceded and/or followed by capital letters and/or
numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted
30 basis, or can be constructed from such available piasmids in accordance with published procedures. In addition,
other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.
"Restriction enzyme digestion" when referring to DNA means catalytic cleavage of internal
phosphodiester bonds of DNA with an enzyme that acts only at certain locations or sites in the DNA sequence.
Such enzymes are called "restriction endonucleases". Each restriction endonuclease recognizes a specific DNA
35 sequence called a "restriction site" that exhibits two-fold symmetry. The various restriction enzymes used
herein are commercially available and their reaction conditions, cofactors, and other requirements as established
by the enzyme suppliers are used. Restriction enzymes commonly are designated by abbreviations composed of
a capital letter followed by other letters representing the microorganism from which each restriction enzyme
originally was obtained and then a number designating the particular enzyme. In general, about 1 ug of plasmid
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or DNA fragment is used with about 1-2 units of enzyme in about 20 ul of buffer solution. Appropriate buffers
and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation of about
1 hour at 37°C is ordinarily used, but may vary in accordance with the supplier's instructions. After incubation,
protein or polypeptide is removed by extraction with phenol and chloroform, and the digested nucleic acid is
5 recovered from the aqueous fraction by precipitation with ethanol. Digestion with a restriction enzyme may be
followed with bacterial alkaline phosphatase hydrolysis of the terminal 5' phosphates to prevent the two
restriction-cleaved ends of a DNA fragment from "circularizing" or forming a closed loop that would impede
insertion of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not
followed by 5' terminal dephosphorylation. Procedures and reagents for dephosphorylation are conventional as
1 0 described in sections 1 .56- 1 .6 1 of Sambrook et al. , Molecular Cloning: A Laboratory Manual (New York: Cold
Spring Harbor Laboratory Press, 1 989).
"Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of
the digest on polyacrylamide or agarose gel by electrophoresis, identification of the fragment of interest by
comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the
15 gel section containing the desired fragment, and separation of the gel from DNA. This procedure is known
generally. For example, see Lawn et al., Nucleic Acids Res., 9:6103-61 14 (1981), and Goeddel et al.. Nucleic
Acids Res., 8:4057 (1980).
"Southern analysis" or "Southern blotting" is a method by which the presence of DNA sequences in a
restriction endonuclease digest of DNA or DNA-containing composition is confirmed by hybridization to a
20 known, labeled oligonucleotide or DNA fragment. Southern analysis typically involves electrophoretic
separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and
transfer of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a
radiolabeled, biotinylated, or enzyme-labeled probe as described in sections 9.37-9.52 of Sambrook et al.,
supra.
25 "Northern analysis" or "Northern blotting" is a method used to identify RNA sequences that hybridize
to a known probe such as an oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment.
The probe is labeled with a radioisotope such as ^-P, or by biotinylation, or with an enzyme. The RNA to be
analyzed is usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to
nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe, using standard techniques
30 well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., supra.
"Ligation" is the process of forming phosphodiester bonds between two nucleic acid fragments. For
ligation of the two fragments, the ends of the fragments must be compatible with each other. In some cases, the
ends will be directly compatible after endonuclease digestion. However, it may be necessary first to convert the
staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for
35 ligation. For blunting the ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15°C with
about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of the four
deoxyribonucleotide triphosphates. The DNA is then purified by phenol-chloroform extraction and ethanol
precipitation. The DNA fragments that are to be ligated together are put in solution in about equimolar
amounts. The solution will also contain ATP, ligase buffer, and a ligase such as T4 DNA ligase at about 10
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units per 0.5 ug of DNA. If the DNA is to be iigated into a vector, the vector is First linearized by digestion
with the appropriate restriction endonuclease(s). The linearized fragment is then treated with bacterial alkaline
phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.
"Preparation" of DNA from cells means isolating the plasm id DNA from a culture of the host cells.
5 Commonly used methods for DNA preparation are the large- and small-scale plasmid preparations described in
sections 1.25-1.33 of Sambrook et ai, supra. After preparation of the DNA, it can be purified by methods well
known in the art such as that described in section 1 .40 of Sambrook et at., supra.
"Oligonucleotides" are short-length, single- or double-stranded polydeoxynucleotides that are
chemically synthesized by known methods (such as phosphotriester, phosphite, or phosphoramidite chemistry,
10 using solid-phase techniques such as described in EP 266,032 published 4 May 1988, or via deoxynucleoside
H-phosphonate intermediates as described by Froehler et ai, Nucl. Acids Res., 14:5399-5407 (1986)). Further
methods include the polymerase chain reaction defined below and other autoprimer methods and
oligonucleotide syntheses on solid supports. All of these methods are described in Engeis et a!., Agnew. Chem.
Int. Ed. Engl.. 28:716-734 (1989). These methods are used if the entire nucleic acid sequence of the gene is
1 5 known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if
the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and
preferred coding residues for each amino acid residue. The oligonucleotides are then purified on
polyacrylamide gels.
"Polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of
20 a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Patent No. 4,683,195
issued 28 July 1987. Generally, sequence information from the ends of the region of interest or beyond needs to
be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in
sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers
may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences,
25 specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA,
bacteriophage or plasmid sequences, etc. See generally Mullis et ai, Cold Spring Harbor Symp. Quant. Biol.
5 1 :263 (1987); Erlich, ed., PCR Technology. (Stockton Press, NY, 1989). As used herein, PCR is considered to
be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid
test sample comprising the use of a known nucleic acid as a primer and a nucleic acid polymerase to amplify or
30 generate a specific piece of nucleic acid.
"Native antibodies and immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000
daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is
linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between
the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced
35 intrachain disulfide bridges. Each heavy chain has at one end a variable domain (Vj|) followed by a number of
constant domains. Each light chain has a variable domain at one and (Vl) and a constant domain at its other
end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the
light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid
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residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al., J.
Mol. Biol., 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-4596 (1985)).
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in
sequence among antibodies and are used in the binding and specificity of each particular antibody for its
5 particular antigen. However, the variability is not evenly distributed through the variable domains of
antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or
hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved
portions of variable domains are called the framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs,
1 0 which form loops connecting, and in some cases forming part of, the b-sheet structure. The CDRs in each chain
are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to
the formation of the antigen binding site of antibodies (see Kabat et al, Sequences of Proteins of
Immunological Interest, National Institute of Health, Bethesda, MD (1987)). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as
1 5 participation of the antibody in antibody-dependent cellular toxicity.
Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab"
fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflects its ability
to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is
still capable of cross-linking antigen.
20 "Fv" is the minimum antibody fragment which contains a complete antigen recognition and binding
site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent
association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen
binding site on the surface of the V^-Vi^ dimer. Collectively, the six CDRs confer antigen binding specificity
to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific
25 for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding
site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain
(CHI) of the heavy chain. Fab" fragments differ from Fab fragments by the addition of a few residues at the
carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge
30 region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear
a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have
hinge cysteines between them. Other, chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one
of two clearly distinct types, called kappa and lambda (I), based on the amino acid sequences of their constant
35 domains.
Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins
can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and
IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-
4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the different classes of
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immunoglobulins are called alpha, delta, epsiion, gamma, and u, respectively. The subunit structures and three-
dimensional configurations of different classes of immunoglobulins are well known.
The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies
(including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as
antibody fragments (e.g., Fab, F(ab'>2, scFv and Fv), so long as they exhibit the desired biological activity.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of
substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies
are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In
addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the
hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is
not to be construed as requiring production of the antibody by any particular method. For example, the
monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma
method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA
methods (see, e.g., U.S. Patent No. 4,816,567 (Cabilly et al.)).
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in
which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived
from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies,
so long as they exhibit the desired biological activity, e.g. binding to and activating mpl (U.S. Patent No.
4,816,567 (Cabilly et at.); and Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For
the most pan, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-
human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibody may comprise residues which are found
neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are
made to further refine and optimize antibody performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise
at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For
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further details see: Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
"Single-chain Fv" or "sFv" antibody fragments comprise the V H and V L domains of antibody, wherein
these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a
5 polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for
antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13,
Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which
fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the
1 0 same polypeptide chain (V H and V L ). By using a linker that is too short to allow pairing between the two
domains on the same chain, the domains are forced to pair with the complementary domains of another chain
and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO
93/11 161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).
The expression "linear antibodies" when used throughout this application refers to the antibodies
15 described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of
tandem Fd segments (V H -C H 1-V H -C H 1) which form a pair of antigen binding regions. Linear antibodies can
be bispecific or monospecific.
A "variant" antibody, refers herein to a molecule which differs in amino acid sequence from a "parent"
antibody amino acid sequence by virtue of addition, deletion and/or substitution of one or more amino acid
20 residue(s) in the parent antibody sequence. In the preferred embodiment, the variant comprises one or more
amino acid substitution(s) in one or more hypervariable region(s) of the parent antibody. For example, the
variant may comprise at least one, e.g. from about one to about ten, and preferably from about two to about
Five, substitutions in one or more hypervariable regions of the parent antibody. Ordinarily, the variant will have
an amino acid sequence having at least 75% amino acid sequence identity with the parent antibody heavy or
25 light chain variable domain sequences, more preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, and most preferably at least 95%. Identity or homology with respect to this sequence is
defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the
parent antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. See Fig. 1 . None of N-terminal, C-terminal, or internal extensions,
30 deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or
homology. The variant retains the ability to bind the receptor and preferably has properties which are superior
to those of the parent antibody. For example, the variant may have a stronger binding affinity, enhanced ability
to activate the receptor, etc. To analyze such properties, one should compare a Fab form of the variant to a Fab
form of the parent antibody or a full length form of the variant to a full length form of the parent antibody, for
35 example, since it has been found that the format of the antibody impacts its activity in the biological activity
assays disclosed herein. The variant antibody of particular interest herein is one which displays at least about
10 fold, preferably at least about 20 fold, and most preferably at least about 50 fold, enhancement in biological
activity when compared to the parent antibody.
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The "parent" antibody herein is one which is encoded by an amino acid sequence used for the
preparation of the variant. Preferably, the parent antibody has a human framework region and has human
antibody constant region(s). For example, the parent antibody may be a humanized or human antibody.
An "isolated" antibody is one which has been identified and separated and/or recovered from a
5 component of its natural environment. Contaminant components of its natural environment are materials which
would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and
other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to
greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid
10 sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the
antibody in situ within recombinant cells since at least one component of the antibody's natural environment
will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
The term "epitope tagged" when used herein refers to an antibody fused to an "epitope tag". The
15 epitope tag polypeptide has enough residues to provide an epitope against which an antibody thereagainst can
be made, yet is short enough such that it does not interfere with activity of the antibody. The epitope tag
preferably is sufficiently unique so that the antibody thereagainst does not substantially cross-react with other
epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-
50 amino acid residues (preferably between about 9-30 residues). Examples include the flu HA tag polypeptide
20 and its antibody 12CA5 (Field et al. Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7,
6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5(12):36 10-36 16 (1985)); and the
Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3(6):547-
553 (1990)). In certain embodiments, the epitope tag is a "salvage receptor binding epitope''. As used herein,
the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule {e.g., IgGi,
25 lgG2, IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
The terms "'mpl ligand", mpl ligand polypeptide", "ML", "thrombopoietin" or "TPO" are used
interchangeably herein and include any polypeptide that possesses the property of binding to mpl, a member of
the cytokine receptor superfamily, and having a biological property of mpl ligand. An exemplary biological
3
property is the ability to stimulate the incorporation of labeled nucleotides (e.g. H-thymidine) into the DNA of
30 IL-3 dependent Ba/F3 cells transfected with human mpl. Another exemplary biological property is the ability to
stimulate the incorporation of 35 S into circulating platelets in a mouse platelet rebound assay. This definition
encompasses a polypeptide isolated from a mpl ligand source such as aplastic porcine plasma described herein
or from another source, such as another animal species, including humans, or prepared by recombinant or
synthetic methods. Examples include TPO(332) and rhTP0 33 2- Also included in this definition is the
35 thrombopoietic ligand described in WO 95/28907 having a molecular weight of about 3 1 ,000 daltons (3 1 kd) as
determined by SDS gel under reducing conditions and 28,000 daltons (28kd) under non-reducing conditions.
The term "TPO" includes variant forms, such as fragments, alleles, isoforms, analogues, chimera thereof and
mixtures of these forms. For convenience, all of these ligands will be referred to below simply as "TPO"
recognizing that all individual ligands and ligand mixtures are referred to by this term.
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Preferably, the TPO is a compound having thrombopoietic activity or being capable of increasing
serum platelet counts in a mammal. The TPO is preferably capable of increasing endogenous platelet counts by
at least 10%, more preferably by 50%, and most preferably capable of elevating platelet counts in a human to
9
greater than about 1 50 X 10 per liter of blood.
5 The TPO of this invention preferably has at least 70% overall sequence identity with the amino acid
sequence of the highly purified substantially homogeneous porcine mpl ligand polypeptide and at least 80%
sequence identity with the "EPO-domain" of the porcine mpl ligand polypeptide. Alternatively, the TPO of this
invention may be a mature human mpl ligand (hML), or a variant or post-transcriptionally modified form
thereof or a protein having about 80% sequence identity with mature human mpl ligand. Alternatively, the TPO
10 may be a fragment, especially an amino-terminus or "EPO-domain" fragment, of the mature human mpl ligand.
Preferably, the amino terminus fragment retains substantially all of the human ML sequence between the first
and fourth cysteine residues but may contain substantial additions, deletions or substitutions outside that region.
According to this embodiment, the fragment polypeptide may be represented by the formula:
X-hTPO(7-151)-Y
15 Where hTPO(7-151) represents the human TPO (hML) amino acid sequence from Cys through
Cys 151 inclusive; X represents the amino group of Cys 7 or one or more of the amino-terminus amino acid
residue(s) of the mature TPO or amino acid residue extensions thereto such as Met, Lys, Tyr or amino acid
substitutions thereof such as arginine to lysine or leader sequences containing, for example, proteolytic cleavage
sites (e.g. Factor Xa or thrombin); and Y represents the carboxy terminal group of Cys 151 or one or more
20 carboxy-terminus amino acid residue(s) of the mature TPO or extensions thereto.
A "TPO fragment" means a portion of a naturally occurring mature full length mpl ligand or TPO
sequence having one or more amino acid residues or carbohydrate units deleted. The deleted amino acid
residue(s) may occur anywhere in the peptide including at either the N-terminal or C-terminal end or internally,
so long as the fragment shares at least one biological property in common with mpl ligand. Mpl ligand
25 fragments typically will have a consecutive sequence of at least 10, 15, 20, 25, 30 or 40 amino acid residues that
are identical to the sequences of the mpl ligand isolated from a mammal including the ligand isolated from
aplastic porcine plasma or the human or murine ligand, especially the EPO-domain thereof. Representative
examples of N-terminal fragments are TPO(153), hML 153 or TPO(Met"' 1-153).
The terms "TPO isoform(s)" and "TPO sequence isoform(s)" or the term "derivatives" in association
30 with TPO, etc. as used herein means a biologically active material as defined below having less than 100%
sequence identity with the TPO isolated from recombinant cell culture, aplastic porcine plasma or the human
mpl ligand. Ordinarily, a biologically active mpl ligand or TPO isoform will have an amino acid sequence
having at least about 70% amino acid sequence identity with the mpl ligand/TPO isolated from aplastic porcine
plasma or the mature murine, human mpl ligand or fragments thereof, preferably at least about 75%, more
35 preferably at least about 80%, still more preferably at least about 85%, even more preferably at least about 90%,
and most preferably at least about 95%.
TPO "analogues" include covalent modification of TPO or mpl ligand by linking the TPO polypeptide
to one of a variety of nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
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4,791,192 or 4,179,337. TPO polypeptides covalently linked to the forgoing polymers are referred to herein as
pegylated TPO.
Still other preferred TPO polypeptides of this invention include mpl ligand sequence variants and
chimeras. Ordinarily, preferred mpl ligand sequence variants and chimeras are biologically active mpl ligand
5 variants that have an amino acid sequence having at least 90% amino acid sequence identity with the human
mpl ligand and most preferably at least 95%. An exemplary preferred mpl ligand variant is a N-terminal
domain hML variant (referred to as the "EPO-domain" because of its sequence homology to erythropoietin).
The preferred hML EPO-domain comprises about the First 153 amino acid residues of mature hML and is
referred to as hML 153. An optionally preferred hML sequence variant comprises one in which one or more of
10 the basic or dibasic amino acid residue(s) in the C-terminal domain is substituted with a non-basic amino acid
residue(s) (e.g., hydrophobic, neutral, acidic, aromatic, Gly, Pro and the like). A preferred hML C-terminal
domain sequence variant comprises one in which Arg residues 153 and 154 are replaced with Ala residues.
This variant is referred to as hML332(R153A, R154A).
A preferred chimera is a fusion between mpl ligand or fragment (defined below) thereof with a
15 heterologous polypeptide or fragment thereof. For example, hML 153 may be fused to an IgG fragment to
improve serum half-life or to IL-3, G-CSF or EPO to produce a molecule with enhanced thrombopoietic or
chimeric hematopoietic activity.
Other preferred mpl ligand fragments have a Met preceding the amino terminus Ser (e.g.
Met"'TPOi53). This is preferred when, for example, the protein is expressed directly in a microorganism such
20 as E coli. Optionally, these mpl ligand fragments may contain amino acid substitutions to facilitate
derivitization. For example, Argi53 or other residues of the carbohydrate domain may be substituted with Lys
to create additional sites to add polyethylene glycol. Preferred mpl ligand fragments according to this option
include Met" 1 TPO(l-X) where X is about 153, 164, 191, 199, 205, 207, 217, 229, or 245 for the sequence of
residues 1-X. Other preferred mpl ligand fragments include those produced as a result of chemical or enzymatic
25 hydrolysis or digestion of the purified ligand.
"Essentially pure" protein means a composition purified to remove contaminating proteins and other
cellular components, preferably comprising at least about 90% by weight of the protein, based on total weight
of the composition, more preferably at least about 95% by weight. "Essentially homogeneous" protein means a
composition comprising at least about 99% by weight of protein, based on total weight of the composition.
30 IL Preferred Embodiments of the Invention
In one embodiment, preferred antibodies of this invention are substantially homogeneous antibodies
and variants thereof, referred to as agonist antibodies (aAb), that possess the property of binding to c-mpl, a
member of the hematopoietic growth factor receptor superfamily, and transducing a survival, proliferation,
maturation and/or differentiation signal. Such signal transduction may be determined by measuring stimulation
35 of incorporation of labeled nucleotides (^H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected
with human mpl P, or with a CMK Assay measuring Induction of the platelet antigen GPIIbIIl a expression.
Signal transduction may also be determined by KIRA ELISA by measuring phosphorylation of the c-mpl-
Rse.gD chimeric receptor, in a c-mpl/Mab HU-03 cell proliferation assay or in a liquid suspension
megakaryocytopoiesis assay .
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Preferred c-mpl agonist antibodies of this invention are also capable of inducing or causing survival,
proliferation, maturation or differentiation of CD34+ cells into the platelet producing form at a concentration
equal to or not less than 2 orders of magnitude (100-fold) below that of thrombopoietin on a weight basis.
More preferred c-mpl aAb(s) are substantially purified aAb(s) having hematopoietic, especially
5 megakaryocytopoietic or thrombocytopoietic activity - namely, being capable of stimulating proliferation,
maturation and/or differentiation of immature megakaryocytes or their predecessors into the mature platelet-
producing form that demonstrate a biological activity equal to or within 2 orders of magnitude of that of rhTPO
on a weight basis. Most preferred aAb(s) of this invention are human aAb(s) including full length antibodies
having an intact human Fc region and including fragments thereof having hematopoietic, megakaryocytopoietic
10 or thrombopoietic activity. Exemplary fragments having the above described biological activity include; Fv,
scFv, F(ab'), F(ab')2.
Preferred scFv fragments denominated 10F6, 5E5, 10D10, 12B5, 12D5 and 12E10 having sequences
for CDRs and Framework regions provided in Figure 1. Alternatively, the above enumerated scFvs are affinity
matured by mutating 1-3 amino acid residues in one or more of the CDRs or in the framework regions between
15 the CDRs.
The framework regions may be derived from a "consensus sequence" (i.e. the most common amino
acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) or may be derived
from an individual human antibody framework region or from a combination of different framework region
sequences. Many human antibody framework region sequences are compiled in Rabat et ai, Sequences of
20 Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
(1991), pages 647-669), for example.
A suitable method for purifying mpl antibodies comprises contacting an antibody source containing the
mpl antibody molecules with an immobilized receptor polypeptide, specifically mpl or a mpl fusion polypeptide,
under conditions whereby the mpl antibody molecules to be purified are selectively adsorbed onto the
25 immobilized receptor polypeptide, washing the immobilized support to remove non-adsorbed material, and
eluting the molecules to be purified from the immobilized receptor polypeptide with an eiution buffer. The
source containing the mpl antibody may be a library of antibodies having different binding epitopes and the
receptor may be immobilized on a plate, tube, particle or other suitable surface using known methods.
Alternatively, the source containing the antibody is recombinant cell culture where the concentration
30 of antibody in either the culture medium or in cell lysates is generally higher than in plasma or other natural
sources. The preferred purification method to provide substantially homogeneous antibody comprises:
removing particulate debris, either host cells or lysed fragments by, for example, centrifugation or
ultrafiltration; optionally, protein may be concentrated with a commercially available protein concentration
filter; followed by separating the antibody from other impurities by one or more steps selected from;
35 immunoaffinity, ion-exchange (e.g., DEAE or matrices containing carboxymethyl or sulfopropyl groups), Blue-
SEPHAROSE, CM Blue-SEPHAROSE, MONO-Q, MONO-S, lentil lectin-SEPHAROSE, WGA-
SEPHAROSE, Con A-SEPHAROSE, Ether TOYPEARL, Butyl TOYPEARL, Phenyl TOYPEARL, protein A
SEPHAROSE, SDS-PAGE, reverse phase HPLC (e.g.. silica gel with appended aliphatic groups) or
SEPHADEX molecular sieve or size exclusion chromatography, and ethanol or ammonium sulfate
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precipitation. A protease inhibitor such as methylsulfonylfluoride (PMSF) may be included in any of the
foregoing steps to inhibit proteolysis.
Preferably, the isolated antibody is monoclonal (Kohler and Milstein, Nature. 256:495-497 (1975);
Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, Burdon et al., Eds, Volume 13,
5 Elsevier Science Publisrers, Amsterdam (1985); and Huse et al. Science, 246:1275-1281 (1989)). A preferred
mpl antibody is one that binds to mpl receptor with an affinity of at least about 1 0 6 1/mole . More preferably
the antibody binds with an affinity of at least about 10 7 1/mole or even at least 10^ 1/mole. Most preferably,
the antibody is raised against a mpl receptor having one of the above described effector functions. The isolated
antibody capable of binding to the mpl receptor may optionally be fused to a second polypeptide and the
10 antibody or fusion thereof may be used to isolate and purify mpl from a source as described above for
immobilized mpl polypeptide. In a further preferred aspect of this embodiment, the invention provides a
method for detecting the mpl ligand in vitro or in vivo comprising contacting the antibody with a sample,
especially a serum sample, suspected of containing the ligand and detecting if binding has occurred.
The invention also provides an isolated nucleic acid molecule encoding the mpl antibody or fragments
15 thereof, which nucleic acid molecule may be labeled or unlabeled with a detectable moiety, and a nucleic acid
molecule having a sequence that is complementary to, or hybridizes under stringent or moderately stringent
conditions with, a nucleic acid molecule having a sequence encoding a mpl antibody. A preferred mpl
antibody nucleic acid is RNA or DNA that encodes a biologically active human antibody.
In a further preferred embodiment of this invention, the nucleic acid molecule is cDNA encoding the
20 mpl antibody and further comprises a replicable vector in which the cDNA is operably linked to control
sequences recognized by a host transformed with the vector. This aspect further includes host cells transformed
with the vector and a method of using the cDNA to effect production of antibody, comprising expressing the
cDNA encoding the antibody in a culture of the transformed host cells and recovering the antibody from the
host cell culture. The antibody prepared in this manner is preferably substantially homogeneous human
25 antibody. A preferred host cell for producing the antibody is Chinese hamster ovary (CHO) cells. An
alternative preferred host cell is E coli.
The invention further includes a preferred method for treating a mammal having an immunological or
hematopoietic disorder, especially thrombocytopenia comprising administering a therapeutically effective
amount of a mpl agonist or antagonist antibody to the mammal. Optionally, the antibody is administered in
30 combination with a cytokine, especially a colony stimulating factor or interleukin. Preferred colony stimulating
factors or interleukins include; kit-ligand, LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, IL-3, IL-5, IL-6,
IL-7, IL-8, 1L-9 or 1L-11. Alternatively, the antibody is administered in combination with an Insulin-like
growth factor (e.g., IGF-1) or a tumor necrosis factor (e.g., lymphotoxin (LT)).
HI. Methods of Making
35 Nucleic acid encoding the agonist and/or antagonist antibodies of the invention can be prepared from
a library of single chain antibodies displayed on a bacteriophage. The preparation of such a library is well
known to one of skill in this art. Suitable libraries may be prepared by the methods described in WO 92/01047,
WO 92/20791, WO 93/06213, WO 93/1 1236, WO 93/19172, WO 95/01438 and WO 95/15388. In a preferred
embodiment, a library of single chain antibodies (scFv) may be generated from a diverse population of human
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B-cells from human donors. mRNA corresponding to the VH and VL antibody chains is isolated and purified
using standard techniques and reverse transcribed to generate a population of cDNA. After PCR amplification,
DNA coding for single chain antibodies is assembled using a linker, such as Gly 4 Ser, and cloned into suitable
expression vectors. A phage library is then prepared in which the population of single chain antibodies is
5 displayed on the surface of the phage. Suitable methods for preparing phage libraries have been reviewed and
are described in Winter et. al., Annu. Rev. Immunol., 1994, 12:433-55; Soderlind et. al., Immunological
Reviews, 1992, 130:109-123; Hoogenboom, Tibtech February 1997, Vol. 15; Neri et. al.. Cell Biophysics,
1995, 27:47-61, and the references described therein.
The antibodies of the invention having agonist or antagonist properties may be selected by
10 immobilizing a receptor and then panning a library of human scFv prepared as described above using the
immobilized receptor to bind antibody. Griffiths et. al., EMBO-J, 1993, 12:725-734. The specificity and
activity of specific clones can be assessed using known assays. Griffiths et. al.; Clarkson et. al., Nature, 1991,
352:642-648. After a first panning step, one obtains a library of phage containing a plurality of different single
chain antibodies displayed on phage having improved binding to the receptor. Subsequent panning steps
15 provide additional libraries with higher binding affinities. When avidity effects are a problem, monovalent
phage display libraries may be used in which less than 20%, preferably less than 10%, and more preferably less
than 1% of the phage display more than one copy of an antibody on the surface of the phage. Monovalent
display can be accomplished with the use of phagemid and helper phage as described, for example, in Lowman
et. al., Methods: A Companion to Methods in Enzymology, 1991, 3(3):205-216. A preferred phage is M13 and
20 display is preferably as a fusion protein with coat protein 3 as described in Lowman et. al., supra. Other
suitable phage include fl and fd filamentous phage. Fusion protein display with other virus coat proteins is
also known and may be used in this invention. See U.S. 5,223,409.
Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide
changes into the antibody DNA, or by peptide synthesis. Such variants include, for example, deletions from,
25 and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies of the
examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired characteristics. The amino acid changes also
may alter post-translational processes of the humanized or variant antibody, such as changing the number or
position of glycosylation sites.
30 A useful method for identification of certain residues or regions of the antibody that are preferred
locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells
Science, 244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most
preferably alanine or polyalanine) to affect the interaction of the amino acids with the receptor. Those amino
35 acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or
other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to
analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the
target codon or region and the expressed antibody variants are screened for the desired activity.
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Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length
from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of
single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal
methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecule
5 include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the
serum half-life of the antibody.
Another type of variant is an amino acid substitution variant. These variants have at least one amino
acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest
interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also
1 0 contemplated. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions".
If such substitutions result in a change in biological activity, then more substantial changes, denominated
"exemplary substitutions" in Table 2, or as further described below in reference to amino acid classes, may be
introduced and the products screened.
Table 2
15
Original Residue
bxemplary
Substitutions
Preferred
Substitutions
A 1 / A '\
Ala (A)
vat; leu; lie
val
Arg(R)
lys; gin; asn
lys
Asn (N)
gin; his; asp, lys; arg
gin
Asp (U)
glu; asn
glu
Cys (Cj
ser; ala
ser
cm (Q)
asn; glu
asn
Glu (b)
asp; gin
asp
(ily(U)
ala
ala
His (H)
asn; gin; lys; arg
arg
He <1J
leu; val; met; ala; phe;
norleucine
leu
Leu (L)
norleucine; lie; val; met;
ala; phe
ile
Lys (K)
arg; gin; asn
arg
Met (M)
leu; phe; ile
leu
Phe (F)
leu; val; ile; ala; tyr
tyr
Pro(P)
ala
ala
Ser (S)
thr
thr
Thr(T)
ser
ser
Trp(W)
tyr; phe
tyr
Tyr(Y)
tip; phe; thr; ser
phe
Val(V)
He; leu; met; phe; ala;
norleucine
leu
Substantial modifications in the biological properties of the antibody are accomplished by selecting
substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone
in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity
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of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into
groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
5 (3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: tip, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another
10 class.
Any cysteine residue not involved in maintaining the proper conformation of the humanized or variant
antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its
stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
15 A particularly preferred type of substitutional variant involves substituting one or more hypervariable
region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such substitutional variants is affinity maturation
using phage using methods known in the art. Briefly, several hypervariable region sites (e.g. 3-7 sites) are
20 mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are
displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3
packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g.
binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identified hypervariable region residues contributing
25 significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure
of the antigen-antibody complex to identify contact points between the antibody and receptor. Such contact
residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
Once such variants are generated, the panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays may be selected for further development.
30 Another type of amino acid variant of the antibody alters the original glycosylation pattern of the
antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding
one or more glycosylation sites that are not present in the antibody.
Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment
of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-
35 serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of
these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers
to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most
commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
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Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid
sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked
glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine
or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
5 Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a
variety of methods known in the art. These methods include, but are not limited to, isolation from a natural
source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-
mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the antibody.
10 Preferably, the antibodies are prepared by standard recombinant procedures which involve production
of the antibodies by culturing cells transfected to express antibody nucleic acid (typically by transforming the
cells with an expression vector) and recovering the antibody from the cells of cell culture.
The nucleic acid (e.g., cDNA or genomic DNA) encoding mpl antibody selected as described above is
inserted into a repiicable vector for further cloning (amplification of the DNA) or for expression. Many vectors
15 are available, and selection of the appropriate vector will depend on (1) whether it is to be used for DNA
amplification or for DNA expression, (2) the size of the nucleic acid to be inserted into the vector, and (3) the
host cell to be transformed with the vector. Each vector contains various components depending on its function
(amplification of DNA or expression of DNA) and the host cell with which it is compatible. The vector
components generally include, but are not limited to, one or more of the following: a signal sequence, an origin
20 of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination
sequence.
(i) Signal Sequence Component
The mpl antibody of this invention may be expressed not only directly, but also as a fusion with a
heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at
25 the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the
vector, or it may be a part of the mpl antibody DNA that is inserted into the vector. The heterologous signal
sequence selected should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the
host cell. For prokaryotic host cells a prokaryotic signal sequence selected, for example, from the group of the
alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the native
30 signal sequence may be substituted by, e.g., the yeast invertase, alpha factor, or acid phosphatase leaders, the C.
albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646
published 15 November 1990. In mammalian cell expression the native signal sequence (i.e., the mpl ligand
presequence that normally directs secretion of mpl ligand from its native mammalian cells in vivo) is
satisfactory, although other mammalian signal sequences may be suitable, such as signal sequences from other
35 mpl ligand polypeptides or from the same mpl ligand from a different animal species, signal sequences from a
mpl ligand, and signal sequences from secreted polypeptides of the same or related species, as well as viral
secretory leaders, for example, the herpes simplex gD signal.
(ii) Origin of Replication Component
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Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate
in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to
replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously
replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin
5 of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 u plasmid origin is
suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning
vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
Most expression vectors are "shuttle" vectors, i.e., they are capable of replication in at least one class
10 of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E.
coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not
capable of replicating independently of the host cell chromosome.
DNA may also be amplified by insertion into the host genome. This is readily accomplished using
Bacillus species as hosts, for example, by including in the vector a DNA sequence that is complementary to a
15 sequence found in Bacillus genomic DNA. Transfection of Bacillus with this vector results in homologous
recombination with the genome and insertion of antibody DNA. However, the recovery of genomic DNA
encoding antibody is more complex than that of an exogenously replicated vector because restriction enzyme
digestion is required to excise the antibody DNA.
(iii) Selection Gene Component
20 Expression and cloning vectors should contain a selection gene, also termed a selectable marker. This
gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective
culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the
culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c)
25 supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for
Bacilli.
One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are
successfully transformed with a heterologous gene express a protein conferring drug resistance and thus survive
the selection regimen. Examples of such dominant selection use the drugs neomycin (Southern et al., J. Molec.
30 Appl. Genet., 1:327 (1982)) mycophenolic acid (Mulligan et al.. Science, 209:1422 (1980)) or hygromycin
Sugden et al., Mol. Cell. Biol., 5:410-413 (1985)). The three examples given above employ bacterial genes
under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt
(mycophenolic acid), or hygromycin, respectively.
Examples of other suitable selectable markers for mammalian cells are those that enable the
35 identification of cells competent to take up the antibody nucleic acid, such as dihydrofolate reductase (DHFR)
or thymidine kinase. The mammalian cell transformants are placed under selection pressure that only the
transformants are uniquely adapted to survive by virtue of having taken up the marker. Selection pressure is
imposed by culturing the transformants under conditions in which the concentration of selection agent in the
medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that
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encodes antibody. Amplification is the process by which genes in greater demand for the production of a
protein critical for growth are reiterated in tandem within the chromosomes of successive generations of
recombinant cells. Increased quantities of antibody are synthesized from the amplified DNA.
For example, cells transformed with the DHFR selection gene are First identified by culturing all of the
5 transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An
appropriate host eel! when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient
in DHFR activity, prepared and propagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216 (1980). The transformed cells are then exposed to increased levels of Mtx. This leads to the synthesis
of multiple copies of the DHFR gene, and, concomitantly, multiple copies of other DNA comprising the
10 expression vectors, such as the DNA encoding antibody. This amplification technique can be used with any
otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenous DHFR
if, for example, a mutant DHFR gene that is highly resistant to Mtx is employed (EP 1 1 7,060). Alternatively,
host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with
DNA sequences encoding antibody, wild-type DHFR protein, and another selectable marker such as
15 aminoglycoside 3 phosphotransferase (APH) can be selected by cell growth in medium containing a selection
agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Patent No. 4,965,199.
A suitable selection gene for use in yeast is the trp\ gene present in the yeast plasmid YRp7
(Stinchcomb et ai, Nature, 282:39 (1979); Kingsman et ai, Gene, 7:141 (1979); or Tschemper et al, Gene,
20 10:157 (1980)). The trp\ gene provides a selection marker for a mutant strain of yeast lacking the ability to
grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence
of the trp\ lesion in the yeast host cell genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly, Z,eM2-deficient yeast strains (ATCC No.
20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
25 (iv) Promoter Component
Expression and cloning vectors usually contain a promoter that is recognized by the host organism and
is operably linked to the antibody nucleic acid. Promoters are untranslated sequences located upstream (5') to
the start codon of a structural gene (generally within about 1 00 to 1 000 bp) that control the transcription and
translation of particular nucleic acid sequence, such as the antibody nucleic acid sequence, to which they are
30 operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters
are promoters that initiate increased levels of transcription from DNA under their control in response to some
change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. At this time
a large number of promoters recognized by a variety of potential host cells are well known. These promoters
are operably linked to antibody encoding DNA by removing the promoter from the source DNA by restriction
35 enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native antibody
promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of
the antibody DNA. However, heterologous promoters are preferred, as they generally permit greater
transcription and higher yields of expressed antibody as compared to the native promoter.
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Promoters suitable for use with prokaryotic hosts include the B-lactamase and lactose promoter
systems (Chang et ai. Nature, 275:615 (1978); and Goeddel et ai. Nature, 281:544 (1979)), alkaline
phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 (1980) and EP 36,776)
and hybrid promoters such as the tac promoter (deBoer et ai, Proc. Natl. Acad Sci. USA, 80:21-25 (1983)).
5 However, other known bacterial promoters are suitable. Their nucleotide sequences have been published,
thereby enabling a skilled worker operably to ligate them to DNA encoding antibody (Siebenlist et al., Cell,
20:269 (1980)) using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial
systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding antibody
polypeptide.
10 Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region
located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence
found 70 to 80 bases upstream from the start of transcription of many genes is a CXCAAT region where X may
be any nucleotide. At the 3 end of most eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly A tail to the 3 end of the coding sequence. All of these sequences are suitably inserted into
1 5 eukaryotic expression vectors.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-
phosphoglycerate kinase (Hitzeman et ai, J. Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess
et ai, J. Adv. Enzyme Reg., 7:149 (1968); and Holland, Biochemistry, 17:4900 (1978)), such as enolase,
glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,
20 glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage of transcription
controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-
25 phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in Hitzeman et ai, EP 73,657A. Yeast enhancers
also are advantageously used with yeast promoters.
Antibody transcription from vectors in mammalian host cells may be controlled, for example, by
promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504
30 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from
heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock
promoters, and from the promoter normally associated with the antibody sequence, provided such promoters are
compatible with the host cell systems.
35 The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction
fragment that also contains the SV40 viral origin of replication. Fiers et ai, Nature, 273:1 13 (1978); Mulligan
and Berg, Science, 209:1422-1427 (1980); Pavlakis et ai, Proc. Natl. Acad. Sci. USA, 78:7398-7402 (1981).
The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindWl E restriction
fragment. Greenaway et ai. Gene, 18:355-360 (1982). A system for expressing DNA in mammalian hosts
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using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this
system is described in U.S. Patent No. 4,601,978. See also Gray et al, Nature, 295:503-508 (1982) on
expressing cDNA encoding immune interferon in monkey cells; Reyes et al, Nature, 297:598-601 (1982) on
expression of human B-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from
5 herpes simplex virus; Canaani and Berg, Proc. Natl. Acad. Set. USA, 79:5166-5170 (1982) on expression of the
human interferon Bl gene in cultured mouse and rabbit cells; and Gorman et al, Proc. Natl Acad Sci. USA,
79:6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo
fibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus
long terminal repeat as a promoter.
10 (v) Enhancer Element Component
Transcription of a DNA encoding the antibody of this invention by higher eukaryotes is often
increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively
orientation and position independent, having been found 5 (Laiminse/ al, Proc. Natl. Acad. Sci. USA, 78:993
15 (1981)) and 3 (Luskye/ al., Mol. Cell Bio., 3:1 108 (1983)) to the transcription unit, within an intron (Banerji et
al, Cell, 33:729 (1983)), as well as within the coding sequence itself (Osborne et al, Mol. Cell Bio., 4:1293
(1984)). Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-
fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples
include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early
20 promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
See also Yaniv, Nature, 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The
enhancer may be spliced into the vector at a position 5 or 3 to the antibody encoding sequence, but is preferably
located at a site 5 from the promoter.
(vi) Transcription Termination Component
25 Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or
nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5 and,
occasionally 3 untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide
segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding antibody.
30 (vii) Construction and Analysis of Vectors
Construction of suitable vectors containing one or more of the above listed components employs
standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the
form desired to generate the plasmids required.
For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to
35 transform E. coli K12 strain 294 (ATCC No. 31,446) and successful transformants selected by ampicillin or
tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction
endonuclease digestion, and/or sequenced by the method of Messing et al. Nucleic Acids Res., 9:309 (1981) or
by the method of Maxam et al, Methods in Enzymology, 65:499 (1980).
(viii) Transient Expression Vectors
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Particularly useful in the practice of this invention are expression vectors that provide for the transient
expression in mammalian cells of DNA encoding the antibody polypeptide. In general, transient expression
involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell
accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide
5 encoded by the expression vector. Sambrook et ai, supra, pp. 16.17 - 16.22. Transient expression systems,
comprising a suitable expression vector and a host cell, allow for the convenient positive identification of
polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired
biological or physiological properties. Thus, transient expression systems are particularly useful in the
invention for purposes of identifying analogues and variants of antibody polypeptide that have antibody
10 polypeptide biological activity.
(ix) Suitable Exemplary Vertebrate Cell Vectors
Other methods, vectors, and host cells suitable for adaptation to the synthesis of the antibody in
recombinant vertebrate cell culture are described in Gething et al.. Nature, 293:620-625 (1981); Mantei et ai,
Nature, 281:40-46 (1979); Levinson et ai; EP 117,060; and EP 117,058. A particularly useful plasmid for
15 mammalian cell culture expression is pRK5 (EP 307,247 U. S. patent no. 5,258,287) or pSVI6B (PCT
Publication No. WO 91/08291).
Suitable host cells for cloning or expressing the vectors herein are the prokaryote, yeast, or higher
eukaryotic cells described above. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-
positive organisms, for example, E. coli, Bacilli such as B. subtilis, Pseudomonas species such as P. aeruginosa,
20 Salmonella typhimurium, or Serratia marcescans. One preferred E. coli cloning host is E. coli 294 (ATCC No.
31,446), although other strains such as E. coli B, E. coli XI 776 (ATCC No. 31,537), and E. coli W3110 (ATCC
No. 27,325) are suitable. These examples are illustrative rather than limiting. Preferably the host cell should
secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro methods of cloning, e.g., PCR or other
nucleic acid polymerase reactions, are suitable.
25 In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts
for antibody encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly
used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains
are commonly available and useful herein, such as Schizosaccharomyces pombe (Beach and Nurse, Nature,
290:140 (1981); EP 139,383 published 2 May 1985), Kluyveromyces hosts (U.S. Patent No. 4,943,529) such as,
30 e.g., K. lactis (Louvencourt et al., J. Bacteriol., 737 (1983)), K. fragilis, K. bulgaricus, K. thermotolerans, and
K. marxianus, yarrowia (EP 402,226), Pichia pastoris (EP 183,070; Sreekrishna et ai, J. Basic Microbiol.,
28:265-278 (1988)), Candida. Trichoderma reesia (EP 244,234), Neurospora crassa (Case et ai, Proc. Natl.
Acad. Sci. USA, 76:5259-5263 (1979)), and filamentous fungi such as, e.g., Neurospora, Penicilliwn,
Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance
35 et ai, Biochem. Biophys. Res. Commun., 1 12:284-289 (1983); Tilbum et al, Gene, 26:205-221 (1983); Yelton
etai, Proc. Natl. Acad. Sci. USA, 81:1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4:475-479
(1985)).
Suitable host cells for the expression of glycosylated antibody are derived from multicellular
organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any
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higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. Examples of
invertebrate cells include plant and insect cells. 'Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),
Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. See,
5 e.g., Luckow et al., Bio/Technology, 6:47-55 (1988); Miller et al., Genetic Engineering, Setlow et al., eds., Vol.
8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature, 315:592-594 (1985). A variety of viral
strains for transfection are publicly available, e.g., the L- 1 variant of Autographa californica NPV and the Bm-5
strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present
invention, particularly for transfection of Spodoptera frugiperda cells.
10 Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as
hosts. Typically, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium
tumefaciens, which has been previously manipulated to contain the antibody DNA. During incubation of the
plant cell culture with A. tumefaciens, the DNA encoding the antibody is transferred to the plant cell host such
that it is transfected, and will, under appropriate conditions, express the antibody DNA. In addition, regulatory
15 and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and
polyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen., 1:561 (1982). In addition, DNA
segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing
transcription levels of plant-expressible genes in recombinant DNA-containing plant tissue. EP 321,196
published 21 June 1989.
20 However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture
(tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse and
Patterson, editors (1973)). Examples of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK,
25 ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells
(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);
30 mouse mammary tumor (MMT 060562, ATCC CCL51); TRJ cells (Mather et al., Annals N.Y. Acad. Sci.,
383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
Host cells are transfected and preferably transformed with the above-described expression or cloning
vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
35 Transfection refers to the taking up of an expression vector by a host cell whether or not any coding
sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan,
for example, CaPC>4 and electroporation. Successful transfection is generally recognized when any indication
of the operation of this vector occurs within the host cell.
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Transformation means introducing DNA into an organism so that the DNA is replicable, either as an
extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is
done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride,
as described in section 1.82 of Sambrook et al, supra, is generally used for prokaryotes or other cells that
5 contain substantial cell-wall barriers. Infection with Agrobacierium tumefaciens is used for transformation of
certain plant cells, as described by Shaw et al, Gene, 23:3 1 5 (1983) and WO 89/05859 published 29 June 1989.
In addition, plants may be transfected using ultrasound treatment as described in WO 91/00358 published 10
January 1991. For mammalian cells without such cell walls, the calcium phosphate precipitation method of
Graham and van der Eb, Virology, 52:456-457 (1978) is preferred. General aspects of mammalian cell host
10 system transformations have been described by Axel in U.S. Patent No. 4,399,216 issued 16 August 1983.
Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bad.,
130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for
introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used.
Prokaryotic cells used to produce the antibody polypeptide of this invention are cultured in suitable
1 5 media as described generally in Sambrook et al. , supra.
The mammalian host cells used to produce the antibody of this invention may be cultured in a variety
of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for
culturing the host cells. In addition, any of the media described in Ham and Wallace, Meth. Enz., 58:44 (1979),
20 Bames and Sato, Anal. Biochem., 102:255 (1980), U.S. Patent No. 4,767,704; 4,657,866; 4,927,762; or
4,560,655; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985; the disclosures of all of which are
incorporated herein by reference, may be used as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal
growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES),
25 nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin™ drug), trace elements
(defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose
or an equivalent energy source. Any other necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH,
and the like, are those previously used with the host cell selected for expression, and will be apparent to the
30 ordinarily skilled artisan.
The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are
within a host animal.
Gene amplification and/or expression may be measured in a sample directly, for example, by
conventional Southern blotting, northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.
35 Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an
appropriately labeled probe, based on the sequences provided herein. Various labels may be employed, most
commonly radioisotopes, particularly 32 P. However, other techniques may also be employed, such as using
biotin-modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding
to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers,
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enzymes, or the like. Alternatively, antibodies may be employed that can recognize specific duplexes, including
DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in
turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the
formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
5 Gene expression, alternatively, may be measured by immunological methods, such as
immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly
the expression of gene product. With immunohistochemical staining techniques, a cell sample is prepared,
typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product
coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels,
10 luminescent labels, and the like. A particularly sensitive staining technique suitable for use in the present
invention is described by Hsu et al.,Am. J. Clin. Path., 75:734-738 (1980).
Antibody preferably is recovered from the culture medium as a secreted polypeptide, although it also
may be recovered from host cell lysates when directly expressed without a secretory signal.
When the antibody is expressed in a recombinant cell other than one of human origin, the antibody is
15 completely free of proteins or polypeptides of human origin. However, it is still usually necessary to purify the
antibody from other recombinant cell proteins or polypeptides to obtain preparations that are substantially
homogeneous as to the mpl ligand per se. As a first step, the culture medium or lysate is centrifuged to remove
particulate cell debris. The membrane and soluble protein fractions are then separated. Alternatively, a
commercially available protein concentration filter (e.g., AMICON or Millipore PELL1CON ultrafiltration
20 units) may be used. The antibody may then be purified from the soluble protein fraction. The antibody
thereafter is purified from contaminant soluble proteins and polypeptides by salting out and exchange or
chromatographic procedures employing various gel matrices. These matrices include; acrylamide, agarose,
dextran, cellulose and others common to protein purification. Exemplary chromatography procedures suitable
for protein purification include immunoaffinity, receptor affinity (e.g., mpl-XgG or protein A SEPHAROSE),
25 hydrophobic interaction chromatography (HIC) (e.g., ether, butyl, or phenyl Toyopearl), lectin chromatography
(e.g., Con A-SEPHAROSE, lentil-lectin-SEPHAROSE), size exclusion (e.g., SEPHADEX G-75). cation- and
anion-exchange columns (e.g., DEAE or carboxymethyl- and sulfopropyl-cellulose), and reverse-phase high
performance liquid chromatography (RP-HPLC) (see e.g., Urdal et ai, J. Chromatog., 296:171 (1984) where
two sequential RP-HPLC steps are used to purify recombinant human 1L-2). Other purification steps optionally
30 include; ethanol precipitation; ammonium sulfate precipitation; chromatofocusing; preparative SDS-PAGE, and
the like.
Antibody variants in which residues have been deleted, inserted, or substituted are recovered in the
same fashion, taking account of any substantial changes in properties occasioned by the variation. For example,
preparation of a an antibody fusion with another protein or polypeptide, e.g., a bacterial or viral antigen,
35 facilitates purification; an immunoaffinity column containing antibody to the antigen can be used to adsorb the
fusion polypeptide. Immunoaffinity columns such as a rabbit polyclonal anti-antibody column can be
employed to absorb the antibody variant by binding it to at least one remaining immune epitope. Alternatively,
the antibody may be purified by affinity chromatography using a purified receptor-IgG coupled to a
(preferably) immobilized resin such as AFFI-Gel 10 (Bio-Rad, Richmond, CA) or the like, by means well
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known in the art. A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to
inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of
adventitious contaminants. One skilled in the art will appreciate that purification methods suitable for native
the antibody may require modification to account for changes in the character of the antibody or its variants
5 upon expression in recombinant cell culture.
In a most preferred embodiment of the invention, the antibodies are agonist antibodies (aAb). By
"agonist antibody" is meant an antibody which is able to bind to and to activate, a particular hematopoietic
growth factor receptor. For example, the agonist may bind to the extracellular domain of the receptor and
thereby cause differentiation and proliferation of megakaryocyte colonies in semisolid cultures and single
10 megakaryocytes in liquid suspension cultures and platelet production in vitro and/or in vivo. The agonist
antibodies are preferably against epitopes within the extracellular domain of the receptor Accordingly, the
antibody preferably binds to substantially the same epitope as the 12E10, 12B5, 10F6, and 12D5 monoclonal
antibodies specifically disclosed herein. Most preferably, the antibody will also have substantially the same or
greater antigen binding affinity as the monoclonal antibodies disclosed herein. To determine whether a
1 5 monoclonal antibody has the same specificity as an antibody specifically disclosed, one can, for example, use a
competitive ELISA binding assay.
DNA encoding the monoclonal antibodies useful in the method of the invention is readily isolated and
sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of human antibodies). The phage of the invention
20 serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors,
which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)
cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells.
IV. Utility
25 The antibodies disclosed herein are useful for in vitro diagnostic assays for activating the receptor of
interest. This is useful in order to study the role of the receptor in megakaryocyte growth and/or differentiation
and platelet production.
The biologically active c-mpl agonist antibody capable of stimulating either proliferation,
differentiation or maturation and/or modulation (either stimulation or inhibition) of apoptosis of hematopoietic
30 cells may be used in a sterile pharmaceutical preparation or formulation to stimulate megakaryocytopoietic or
thrombopoietic activity in patients suffering from thrombocytopenia due to impaired production, sequestration,
or increased destruction of platelets. Thrombocytopenia-associated bone marrow hypoplasia (e.g., aplastic
anemia following chemotherapy or bone marrow transplant) may be effectively treated with the aAb
compounds of this invention as well as disorders such as disseminated intravascular coagulation (DIC), immune
35 thrombocytopenia (including HIV-induced ITP and non HIV-induced ITP), chronic idiopathic
thrombocytopenia, congenital thrombocytopenia, myelodysplasia, and thrombotic thrombocytopenia.
Preferred uses of the megakaryocytopoietic or thrombocytopoietic biologically active c-mpl agonist
antibody of this invention are in: myelotoxic chemotherapy for treatment of leukemia or solid tumors,
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myeloablative chemotherapy for autologous or allogeneic bone marrow transplant, myelodysplasia, idiopathic
aplastic anemia, congenital thrombocytopenia, and immune thrombocytopenia.
The biologically active c-mpl agonist antibody of the instant invention may be employed alone or in
combination with other cytokines, hematopoietins, interleukins, growth factors, or antibodies in the treatment of
5 the above-identified disorders and conditions. Thus, the instant compounds may be employed in combination
with other protein or peptide having hematopoietic activity including G-CSF, GM-CSF, L1F, M-CSF, IL-1, IL-
3, erythropoietin (EPO), kit ligand, IL-6, and IL-1 1.
The biologically active c-mpl agonist antibody of the instant invention may be used in the same way
and for the same indications as thrombopoietin (TPO). Some forms of the aAb have a longer half-life than
10 native or pegylated TPO and thus are used in indications where a longer half-life are indicated.
When used for in vivo administration, the antibody formulation must be sterile. This is readily
accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and
reconstitution. The antibody ordinarily will be stored in lyophilized form or in solution.
Therapeutic antibody compositions generally are placed into a container having a sterile access port,
1 5 for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
The route of antibody administration is in accord with known methods, e.g., injection or infusion by
intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intrathecal, inhalation or
intralcsional routes, or by sustained release systems as noted below. The antibody is preferably administered
continuously by infusion or by bolus injection.
20 An effective amount of antibody to be employed therapeutically will depend, for example, upon the
therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be
necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the
optimal therapeutic effect. Typically, the clinician will administer antibody until a dosage is reached that
achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
25 The antibodies of the invention may be prepared in a mixture with a pharmaceutically acceptable
carrier. This therapeutic composition can be administered intravenously or through the nose or lung, preferably
as a liquid or powder aerosol (lyophilized). The composition may also be administered parenterally or
subcutaneously as desired. When administered systematically, the therapeutic composition should be sterile,
pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability.
30 These conditions are known to those skilled in the art. Briefly, dosage formulations of the compounds of the
present invention are prepared for storage or administration by mixing the compound having the desired degree
of purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are non-toxic to the
recipients at the dosages and concentrations employed, and include buffers such as TRJS HC1, phosphate,
citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than
35 about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic
acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or
its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol
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or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS or
polyethyleneglycol.
Sterile compositions for injection can be formulated according to conventional pharmaceutical
practice. For example, dissolution or suspension of the active compound in a vehicle such as water or naturally
5 occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the
like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted
pharmaceutical practice.
Suitable examples of sustained-release preparations include semipermeable matrices of solid
hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films,
10 or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-
hydroxyethyl-methacrylate) as described by Langer et ai, J. Biomed. Mater. Res., 15:167-277 (1981) and
Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et ai, Biopolymers, 22:547-556
(1983)), non-degradable ethylene-vinyl acetate (Langer et a/., supra), degradable lactic acid-glycolic acid
15 copolymers such as the LUPRON Depot™ (injectable microspheres composed of lactic acid-glycolic acid
copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules
for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins
remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C,
20 resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be
devised for protein stabilization depending on the mechanism involved. For example, if the aggregation
mechanism is discovered to be intermolecular S-S bond formation through disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific polymer matrix compositions.
25 Sustained-release compositions also include liposomally entrapped TPO. Liposomes containing TPO
are prepared by methods known per se: DE 3,218,121; Epstein et ai, Proc. Natl. Acad. Sci. USA. 82:3688-
3692 (1985); Hwang et ai, Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP 36,676; EP
88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent Nos. 4,485,045 and
4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamellar
30 type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being
adjusted for the optimal therapy.
The dosage of the antibody will be determined by the attending physician taking into consideration
various factors known to modify the action of drugs including severity and type of disease, body weight, sex,
diet, time and route of administration, other medications and other relevant clinical factors. Therapeutically
35 effective dosages may be determined by either in vitro or in vivo methods.
An effective amount of the agonist antibody to be employed therapeutically will depend, for example,
upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it
will be necessary for the therapist to titer the dosage and modify the route of administration as required to
obtain the optimal therapeutic effect. A typical daily dosage might range from about 1 Hg/kg to up to 1000
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mg/kg or more, depending on the factors mentioned above. Typically, the clinician will administer the
molecule until a dosage is reached that achieves the desired effect. The progress of this therapy is easily
monitored by conventional assays.
Depending on the type and severity of the disease, from about 0.00 1 mg/kg to about 1 000 mg/kg, more
5 preferably about 0.01 rag to 100 mg/kg, more preferably about 0.010 to 20 mg/kg of the agonist antibody might
be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. For repeated administrations over several days or longer, depending
on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs or the desired
improvement in the patient's condition is achieved. However, other dosage regimens may also be useful.
10 Examples
Without further description, it is believed that one of ordinary skill in the art can, using the preceding
description and illustrative examples, make and utilize the present invention to the fullest extent. The following
working examples therefore specifically point out preferred embodiments of the present invention, and are not
to be construed as limiting in any way of the remainder of the disclosure.
15 Example 1
Assays
The mpl agonist antibody assays were conducted essentially as described in WO 95/18858.
(a) Ba/F3 cell proliferation assay
The Ba/F3-mpl cell line was established (F. de Sauvage et al., Nature, 369:533 (1994)) by introduction
20 of the cDNA encoding the entire mpl receptor into the IL-3 dependent murine lymphoblastoid cell line Ba/F3.
Stimulation of proliferation of Ba/F3-mpl cells in response to various concentrations of antibodies or TPO was
measured by the amount of incorporation of ^H-thymidine as previously described (F. de Sauvage et al,
supra).
(b) CMK Assay for Induction of Platelet Antigen GPII h HI a Expression
25 CMK cells are maintained in RMPI 1640 medium (Sigma) supplemented with 10% fetal bovine serum
and lOmM glutamine. In preparation for the assay, the cells are harvested, washed and resuspended at 5x10^
cells/ml in serum-free GIF medium supplemented with 5mg/l bovine insulin, 10mg/l apo-transferrin, 1 X trace
elements. In a 96-well flat-bottom plate, the TPO standard or experimental agonist antibody samples are added
to each well at appropriate dilutions in 100 ml volumes. 100 ml of the CMK cell suspension is added to each
30 well and the plates are incubated at 37°C, in a 5% CO2 incubator for 48 hours. After incubation, the plates are
spun at lOOOrpm at 4°C for five minutes. Supernatants are discarded and 100 ml of the FITC -conjugated
GPIl D III a monoclonal 2D2 antibody is added to each well. Following incubation at 4°C for 1 hour, plates are
spun again at lOOOrpm for five minutes. The supernatants containing unbound antibody are discarded and 200
ml of 0.1% BSA-PBS wash is added to each well. The 0.1% BSA-PBS wash step is repeated three times. Cells
35 are then analyzed on a FASCAN using standard one parameter analysis measuring relative fluorescence
intensity.
(c) KIRA EL1SA for Measuring Phosphorylation of the mpl-Rse.gD Chimeric Receptor
The human mpl receptor has been disclosed by Vigon et al., PNAS, USA 89:5640-5644 (1992). A
chimeric receptor comprising the extracellular domain (ECD) of the mpl receptor and the transmembrane and
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intracellular domain (ICD) of Rse (Mark et ai, J. of Biol. Chem. 269(14): 10720-10728 (1994)) with a carboxyl-
terminal flag polypeptide (i.e. Rse.gD) was made for use in the KIRA ELISA described herein.
(i) Capture agent preparation
Monoclonal anti-gD (clone 5B6) was produced against a peptide from Herpes simplex virus
5 glycoprotein D (Paborsky el ai, Protein Engineering 3(6):547-553 (1990)). The purified stock preparation was
adjusted to 3.0mg/ml in phosphate buffered saline (PBS), pH 7.4 and 1 .0ml aliquots were stored at -20° C.
(ii) Anti-phosphotyrosine antibody preparation
Monoclonal anti-phosphotyrosine, clone 4G10, was purchased from UBI (Lake Placid, NY) and
biotinylated using long-arm biotin-N-hydroxysuccinamide (Biotin-X-NHS, Research Organics, Cleveland,
10 OH).
(iii) Ligand
The mpl ligand was prepared by the recombinant techniques described herein. The purified mpl
ligand was stored at 4 °C. as a stock solution.
KIRA ELISA results for agonist antibodies of the invention are shown in Fig. 9. This assay indicates
15 that the antibodies of the invention activate the mpl receptor to a degree similar to the cognate ligand TPO.
(d) TPO receptor-binding inhibition assay
NUNC 96-well immunoplates were coated with 50 ul of rabbit anti-human IgG Fc (Jackson
Labs) at 2 ug/ml in carbonate buffer (pH9.6) overnight at 4°C. After blocking with ELISA buffer (PBS, 1 %
BSA, 0.2 % TWEEN 20), the plates were incubated for 2 hr with conditioned media from mpl-Ig-transfected
20 293 cells. Plates were washed, and 2.5 ng/ml biotinylated TPO was added in the presence or absence of
various concentrations of antibodies. After incubation for 1 hr and washing, the amount of TPO bound was
detected by incubation with streptAvidin-HRP (Sigma) followed by TMB peroxidase substrate (Kirkegaard &
Perry). All dilutions were performed in ELISA buffer, and all incubations were at room temperature. Color
development was quenched with H3PO4 and absorbance was read at 450-650 nm.
25 £e) HU-03 cell proliferation assay
The HU-01 cell line (D. Morgan, Hahnemann University) is derived from a patient with acute
megakaryoblastic leukemia and is dependent on granulocyte-macrophage colony stimulating factor (GM-CSF)
for growth. The HU-03 cell line used here was derived from HU-01 cells by adaptation to growth in rhTPO
rather than GM-CSF.
30 HU-03 cells were maintained in RPMI 1640 supplemented with 2 % heat-inactivated human
male serum and 5 ng/ml rhTPO. Before assay, cells were starved by removing TPO, decreasing serum
concentrration to 1 %, and adjusting the concentration of cells to 2.5 x 10 5 cells/ml, followed by incubation for
16 hr. Cells were then washed and seeded into 96-well plates at a density of 5 x 10 4 cells per well in medium
containing TPO or antibodies at various concentrations. Quadruplicate assays were performed. 1 uCi 3H-
35 thymidine was added to each well before incubation for 24 hr. Cells were collected with a Packard cell
harvester and incorporation of ^H-thymidine was measured with a Top Count Counter (Packard).
(f) Liquid suspension megakarvocvtopoeisis assay
The effect of Mpl agonist antibodies on human megakaryocytopoiesis was determined using a
modification of the liquid suspension assay previously described (Grant et al, Blood 69:1334-1339 (1997)).
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Buffy coats were collected from human umbilical cord blood and cells washed in phosphate-buffered saline
(PBS) by centrifugation at 120 g for 15 min at room temperature to remove platelet-rich plasma. Cell pellets
were resuspended in Iscove's modified Dulbecco's medium (IMDM, GIBCO) (supplemented with 100 units per
ml penicillin and streptomycin), layered onto 60% percoll (density = 1.077 gm/ml, Pharmacia), and centrifuged
5 at 800 g for 20 min at room temperature. The light-density mononuclear cells were collected from the
interface and washed twice with IMDM. Cells were seeded at 1 x 10^ cells per ml in IMDM supplemented
with 30% fetal bovine serum (FBS), 100 units per ml penicillin and streptomycin, and 20 uM 2-
mercaptoethanol, into 24-well tissue culture plates (COSTAR). Serial dilutions of thrombopoietin (TPO) or the
Fab'2 forms of antibody 12B5 or antibody 12D5 were added to quadruplicate wells; control wells contained no
10 additional supplements. Final volumes were 1 ml per well. The cultures were grown in a humidified incubator
at 37 °C in 5% CC>2 for 14 days. Megakaryocytopoiesis was quantified using radiolabelled murine monoclonal
antibody HP1-1D (provided by W. L. Nichols, Mayo Clinic) which has been shown to be specific for the
human megakaryocyte glycoprotein Ilb/IIIa (Grant et al., supra). Cells were harvested from the tissue culture
plates, washed twice with assay buffer (20% FBS, 0.002% EDTA in PBS), and resuspended in 100 ul assay
15 buffer containing 20 ng iodinated HP1-1D (approximatedly 100,000 cpm). After incubation at room
temperature for 1 hr, the cells were washed twice with assay buffer and the cell pellets counted with a gamma
counter.
FBS used in this assay was treated with Dextran T40 at 1 mg/ml and charcoal at 10 mg/ml for 30 min,
centrifuged, decanted, filter sterilized and heat inactivated at 56 °C for 30 min.
20 (g) TPO-antibodv competitive binding assays for HU-03 cells and human platelets
HU-03 cells were cultured as described above. Platelet rich plasma (PRP) was prepared by
centrifugation of citrated whole blood at 400 g's for 5 minutes. Binding studies were conducted within three
hours of collection. 125 I-TPO was prepared by indirect iodination (Fielder, P. J., Hass, P., Nagel, M.,
Stefanich, E., Widmer, R., Bennett, G. L., Keller, G., de Savage, F. J., and Eaton, D. 1997. Human platelets as
25 a model for the binding and degradation of thrombopoietin. Blood 89: 2782-2788) and yielded a specific
activity of 15-50 uCi/ug protein.
In a volume of 110 microliters containing lOOpM iodinated TPO, 2 x 10^ washed HU-03 cells in
Hank's Balanced Salt Solution, 5 mg/ml bovine serum albumin (HBSSB), or 4 x 10^ platelets in plasma, were
incubated at 37°C for 30 minutes with varying concentrations of antibody in triplicate. HU-03 cells were
30 agitated during the incubation period to keep them in suspension. The reaction mixture was overlayed on 1 ml
20 % sucrose-HBSSB and microcentrifuged at 13,500 rpm for five minutes. The supernatants were aspirated,
tube bottoms containing the cell pellets were cut off, and cell- or platelet-associated radioactivity was measured
with an Iso Data Model 120 gamma counter.
Results for several agonist antibodies on the invention in this assay are shown in Fig. 10A-F. Longer
35 bars in the graphs indicate greater amounts of bound radiolabeled TPO and less competition by the agonist
antibody at a particular concentration.
(h) Affinity determinations .
The receptor-binding affinities of several Fab fragments were calculated (Lofas & Johnsson, 1990)
from association and dissociation rate constants measured using a BIACORE surface plasmon resonance system
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(Pharmacia Biosensor). A biosensor chip was activated for covalent coupling of gD-mpl receptor using N-
ethyl-N"'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS)
according to the supplier's (Pharmacia Biosensor) instructions. gD-mpl was buffer-exchanged into 10 mM
sodium acetate buffer (pH 4.5) and diluted to approximately 30 ug/mL. An aliquot (35 u,L) was injected at a
5 flow rate of 1 uL/min to achieve approximately 6400 response units (RU) of coupled protein. Finally, 1M
ethanolamine was injected as a blocking agent. For kinetics measurements, 1.5 serial dilutions of Fab were
injected in PBS/Tween buffer (0.05% Tween-20 in phosphate buffered saline) at 25°C using a flow rate of 20
uL/min. Equilibrium dissociation constants, K^'s, from SPR measurements were calculated as k 0 ff/k on .
Standard deviations, s on for k on and s 0 ff for k 0 ff, were obtained from measurements with >4 protein
10 concentrations (k on ) or with >7 protein concentrations (k Q ff). Dissociation data were fit to a simple AB— >A+B
model to obtain koff +/- s.d. (standard deviation of measurements). Pseudo-first order rate constant (ks) were
calculated for each association curve, and plotted as a function of protein concentration to obtain kon +/- s.e.
(standard error of fit). The resulting errors e[K] in calculated Kj's were estimated according to the following
formula for propagation of errors: e[K] = [(k on )" (s off ) + (k off ) (k on )~ (s on )^] where s Q ff and s on are
1 5 the standard errors in k 0 n and koff. res P ect ' ve 'y-
Example 2
Isolation of Antibodies from the CAT library
For construction of a library of antibodies displayed of a phage see the following references: WO
92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.
9
20 Briefly, Figures 2 and 3 presents a cartoon of the construction of a phage library containing 6x10 different
clones containing single-chain Fv (scFv) antibodies fused to gene 3 of a phage. Binding selection against an
antigen , in this case c-mpl, can be carried out as shown in Fig. 4 and described in greater detail below.
(a) The Antigen
Human c-mpl was cloned as described by F. de Sauvage et a/., Nature 369:533 (1994).
25 £b) Phage Selection on immunorubes
NUNC immunotubes were coated with 2 ml of a solution of 10 microg/ml of gD-c-rnpl in PBS at 4°C
overnight. After rinsing with PBS, tubes were blocked with 3% dry milk in PBS (MPBS) for 2 hr at room
12
temperature. For the first round, 10 ul of C.A.T. antibody phage library containing -1x10 c.f.u. were added
to 1 ml MPBS for blocking for 1 hr at room temperature. Blocked phage were added to coated tubes, and
30 binding of phage to antigen allowed to continue for 2 hr at 37°C on a rotating wheel. Tubes were washed 6
times with PBS-TWEEN and 6 times with PBS, and phage were then eluted with 100 mM TEA for 10 min at
room temperature, neutralized with 500 ul of 1 M TR1S (pH 7.4), and stored on ice until needed. For
subsequent rounds, washing was increased to 20 times with PBS-TWEEN, and 20 times with PBS.
Eluted phage were used to infect 5 ml of log phase E. coli TGI cells and plated on 2YT agar
35 supplemented with 2% glucose and 100 ug/ml carbenicillin. After overnight growth at 30°C, colonies were
scraped into 10 ml 2YT. 50 ul of this solution was used to inoculate 25 ml of 2YT with carbenicillin and
glucose and incubated, shaking, for two hours at 37°C. Helper phage M13K07 (Pharmacia) were added at an
m.o.i. of 10. After adsorption, the cells were pelleted and resuspended in 25 ml of 2YT with carbenicillin
(lOOug/ml) and kanamycin (50ug/ml) and growth continued at 30°C for 4 hr. E. coli were removed from the
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phage by centrifugation, and 1 ml of these phage (approx. 10 12 c.f.u.) were used in subsequent rounds of
selection.
(c) Antibody Phage Selection using streptavidin-coated paramagnetic beads
The library was also selected using soluble biotinylated antigen and streptavidin-coated paramagnetic
5 beads (see Fig. 5). gD-c-mpl was biotinylated using IMMUNOPURE NHS-biotin (biotiny-N-hydroxy-
succinimide, Pierce) according to manufacturer's recommendations.
For the first round of panning, 10 ul of the phage library were blocked with 1 ml of MPBST (3% dry
milk powder, 1 x PBS, 0.2% TWEEN 20) for 1 hour on a rotating wheel at room temperature. Biotinylated gD-
c-mpl was then added to a final concentration of 100 nM, and phage were allow to bind antigen for 1 hour at
10 37°C on a rotator. Meanwhile, 300 ul of DYNABEADS M-280, coated with streptavidin (DYNAL) were
washed 3 times with 1 ml MPBST (using a DYNAL Magnetic Particle Concentrator) and then blocked for 2 hr
at 37°C with 1 ml fresh MPBST on a rotator. The beads and were collected with the MPC, resuspended in 50
ul of MPBST, and added to the phage-plus-antigen solution. Mixing continued on a wheel at room temperature
for 15 min. The DYNABEADS and attached phage were then washed a total of 7 times: 3 times with 1 ml
15 PBS-TWEEN, once with MPBS, followed by 3 times with PBS. Phage were eluted from the beads by
incubating 5 min at room temperature with 300 ul of 100 mM triethylamine. The phage-containing supernatant
was removed and neutralized with 150 ul of 1M TRIS-HC1 (pH 7.4). Neutralized phage were used to infect
mid-log TG 1 host cells as described above. Plating, induction and harvesting of phage were also as for
selection on tubes.
20 For the second and subsequent rounds of selection on biotinylated gD-c-mpl, 1 ml of harvested phage
(approximately 10 12 cfu) were blocked with 200 ul 10% dry milk, 6 X PBS, 0.3% TWEEN 20. Antigen
concentration was decreased at each round of selection. In one series the concentrations were: first round, 100
nM; second round, 10 nM; third round, 1 nM. A second panning was performed using: first round 100 nM;
second round 100 nM; third round, 50 nM; fourth round, 10 nM; and fifth round, 1 nM. Washing stringency
25 was increased to two cycles of 7 washes for rounds 2, and three cycles for rounds 3 and beyond.
(d) ELISA screening of selected clones
After each round of selection, individual carbenicillin-resistant colonies were screened by ELISA to
identify those producing c-mpl-binding phage. Only those clones which were positive in two or more assay
formats were further studied. Fig. 6 illustrates the phage ELISA process.
30 Individual clones were inoculated into 2TY with 2% glucose and 100 ug/ml carbenicillin in 96-well
tissue culture plates and grown until turbid. Cultures were then infected at an m.o.i. of 10 with M12K07 helper
phage, and infected cells were transferred to 2YT media containing carbenicillin (100 ug/ml) and kanamycin
(50 ug/ml) for growth overnight at 30°C with gentle shaking.
NUNC MAXISORP microtiter plates were coated with 50 ul per well of gD-c-mpl, BSA, or gD-
35 gp 120, at 2 ug/ml in 50 mM carbonate buffer (pH 9.6), at 4°C overnight. After removing antigen, plates were
blocked with 3% dry milk in PBS (MPBS) for 2 hours at room temperature.
Phage cultures were centrifuged and 100 ul of phage-containing supernatants were blocked with 20
ul of 6 x PBS / 18% dry milk for 1 hour at room temperature. Block was removed from titer plates and blocked
phage added and allowed to bind for 1 hour at room temperature. After washing, phage were detected with a
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1:5000 dilution of horseradish peroxidase-conjugated anti-M13 antibody (Pharmacia) in MPBS followed by
S'.S'.S'.S'-tetramethylbenzidine (TMB). Reactions were stopped by the addition of H 2 S0 4 and readings taken
by subtracting the A 405nm from the A 450nm .
(e) Soluble scFv ELISA
5 Soluble scFv was induced in the bacterial supernatants of clones by growth in 2YT containing
carbenicillin (100 ug/ml) and IPTG (ImM) ON at 30°C. ELISA plates were either coated with gD-c-mpl or, for
capture ELISA, with anti-c-myc Mab 9E10. Plates were blocked with 1 x ELISA diluent (PBS supplemented
with 0.5% BSA, 0.05% Tween 20, pH 7.4), and soluble scFv was blocked by adding 20 ul of 6 x ELISA dil to
100 ul of supernatant. After binding to antigen coated plates, soluble scFv was detected by adding 50 ul of 1
10 ug/ml Mab 9E10 per well, followed by horseradish peroxidase-conjugated goat anti-murine Ig, and then TMB
as described above. For capture ELISA, soluble scFv was detected by addition of biotinylated c-mpl, followed
by streptavidin-peroxidase conjugate and then TMB as above.
The number of clones screened by ELISA from each round, and the number of clones positive by
phage ELISA are shown in Table 3.
15 Table 3 - Anti-c-mpl scFv antibodies from CAT library
Clones screened: 1534
Clones positive by ELISA: 361
Clones different by BstNI and sequencing: 24
Clones that express protein well 17
20 clones that are agonists by KIRA: 9
clones that are agonists by BaF3 proliferation assay: 6
clones that are agonists by Hu3 proliferation assay: 4
(f) DNA fingerprinting of clones
The diversity of c-mpl-binding clones was determined by PCR amplifying the scFv insert using
25 primers pUC19R (5 AGC GGA TAA CAA TTT CAC ACA GG 3 ) (SEQ. ID. NO: 54) which anneals upstream
of the leader sequence and fdtetseq (5 GTC GTC TTT CCA GAC GGT AGT 3 ) (SEQ. ID. NO: 55) which
anneals in the 5 end of gene III, followed by digestion with the frequent-cutting restriction enzyme BstNI (see
Fig. 7).
Typical patterns seen after analysis on a 3% agarose gel are shown in Fig. 8A-C.
30 DNA Fingerprinting: Protocol
Mix A: dH20 67 ul
10 x ampliTaq buffer 10
25mMMgC12 10
DMSO, 50% 2
35 forward primer 1
Mix B: 2.5 mM dNTPs 8 ul
AMPLITAQ 0.5
reverse primer 1 .0
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Place 90 \i\ of Mix A in reaction tube
Inoculate with very small portion of E.coli colony using a yellow tip
Heat in PCR block to 98°C, 3 min. Remove to ice.
Add 10 nl Mix B
5 Cycle: 95° C, 30 sec, 55°C 30 sec, 72°C lmin 20 sec, for 25 cycles, in Perkin Elmer 2400
Remove 1 0 ul ro run on a 1 % agarose gel, test for a 1 kB band
Make remaining mix to 1 x BstNI reaction buffer
Add 5 units BstNI
10 60°C, 2 hours
Electrophorese samples on 3 % METAPHORE agarose gel
(g) Sequencing of clones
The nucleotide sequence of representative clones of each fingerprint were obtained. Colonies were
inoculated into 50 ml of LB medium supplemented with 2% glucose and 100 ug/ml carbenicillin, and grown
15 overnight at 30°C. DNA was isolated using Qiagen Tip- 100s and the manufacturer's protocol and cycle
sequenced with fluorescent dideoxy chain terminators (Applied Biosystems). Samples were run on an Applied
Biosystems 373A Automated DNA Sequencer and sequences analyzed using the program "Sequencher" (Gene
Codes Corporation). The VH and VL genes were assigned to a germline segment using the antibody database,
V-BASE.
20 DNA sequence was obtained for 39 clones and resulted in 24 different c-mpl-binding scFvs.
(h) Purification of scFvs with (his) 6
For protein purification of soluble antibody, E. coli strain 33D3 was transformed with phagemid DNA.
Five ml of 2YT with carbenicillin and glucose was used to grow overnight cultures at 30°C. 0.2 ml of these
cultures were diluted into 200 ml of the same media and grown to an OD 600 of approximately 0.9. The cells
25 were pelleted and resuspended in 250 ml of 2YT containing IPTG (1 mM) and carbenicillin (100 ug/ml) and to
induce expression and grown for a further 5 hours at 30°C. Cell pellets were harvested and frozen at -20°C.
The antibodies were purified by immobilized metal chelate affinity chromatography (IMAC). Frozen
pellets were resuspended in 10 ml of ice-cold shockate buffer (25 mM TRIS-HC1, 1 mM EDTA, 200 mM NaCI,
20 % sucrose, 1 mM PMSF) by shaking on ice for 1 hr. Imidazole was added to 20 mM, and cell debris
30 removed by centrifugation. The supernatants were adjusted to ImM MgCl 2 and 50 mM phosphate buffer pH
7.5. Ni-NTA agarose resin from Qiagen was used according to the manufacturers instructions. The resin was
equilibrated with 50 mM sodium phosphate buffer pH 7.5, 500 mM NaCI, 20 mM imidazole, and the shockate
added. Binding occurred in either a batch mode or on a gravity flow column. The resin was then washed twice
with 10 bed volumes of equilibration buffer, and twice with buffer containing imidazole increased to 50mM.
35 Elution of proteins was with 50 mM phosphate buffer pH 7.5, 500 mM NaCI and 250 mM imidazole. Excess
salt and imidazole was removed on a PD-10 column (Pharmacia), and proteins were concentrated using a
Centricom 10 to a volume of about 1 ml.
Concentration was estimated spectrophotometrically assuming an A280 nm of 1.0 = 0.6 mg/ml.
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Portions of these protein preparations were submitted for KIRA assay, c-mpl-Ba/F3 cell proliferation
assay, and Hu3 cell proliferation assay.
Plasmid DNA for scFv clones 12B5, 12D5, 12E10, 10D10, 10F6 and 5E5 (named
pMpl.12B5.scFv.his; pMpl.12D5.scFv.his; pMpl.12E10.scFv.his; pMpl.10D10.scFv.his; pMpl.10F6.scFv.his;
5 and pMpl.5E5.scFv. his, respectively) has been deposited with ATCC, Manassas, Virginia, USA.
£]} Reformatting of antibodies to scFv with gD tag. Fab'. Fab'2. and full length molecules .
For improved expression of scFv, and for Fab', and Fab'2 forms of antibodies, some of the anti-c-mpl
clones were cloned into derivatives of the expression vector pAK19 (Carter et al. METHODS: A companion to
Methods in Enzymology. 3:183-192 (1991). Expression is under the transcriptional control of the E. coli
10 alkaline phosphatase (phoA) promoter (Chang, et al Gene 44:121-125 (1986) which is inducible by phosphate
starvation. Each peptide chain is preceded by the E. coli enterotoxin II (stll) signal sequence (Picken, et al.) to
direct secretion to the periplasmic space of E. coli.. This vector also contains the human kj (Palm et al..
Infect. Immun. 42:269-275 (1983)) and the human IgGl C H 1 (Ellison, et al, Nucleic Acids Res. 10: 4071-4079
(1982)) constant domains. The C^l gene is immediately followed by the bacteriophage X t 0 transcriptional
15 terminator (Scholtissek and Grosse Nucleic Acids Res. 15:3185 (1987)).
(j) Fab' and Fab'2 construction
Construction of the Fab' and Fab'2 variants was facilitated by insertion into pAK19 of unique
restriction sites at the junctions of the stll and V L domain (Sfi I), the V ( and Ck domains (Rsr II), the stll and V H
domain (Mlul), and the V H and C H 1 domains (Apa I), using oligonucleotide directed mutagenesis. In order to
20 insure expression of monovalent Fab' molecules, the free cysteine at the 3' end of the CHI domain was mutated
to a threonine, these Fab' molecules thus end in the amino acid sequence thr-ala-ala-pro, rather than thr-cys-
ala-ala as in pAK19. This vector for the expression of Fab' molecules is named pXCA730.
Since some of the antibodies derived from the library had light chains which were derived from
lambda rather than kappa light chain families, the human X C L was subcloned from pBl 1.2 (Carter, P, Garrard,
25 L., Henner, D. 1991. Methods: A Companion to Methods in Enzymology. 3:183-192) into a derivative of
pXCA730 to give vector pXCA970.
For expression of the antibodies as Fab'2 molecules, a vector was constructed which adds the human
IgGl hinge region onto the C H 1 domain of pXCA730. This is followed by the yeast GCN4 leucine zipper
domain (Hu, et al. Science 250:1400-1403 (1990)) for stability. These DNA fragments were constructed using
30 synthesized oligonucleotides and encode the amino acid sequence: cys-pro-pro-cys-ala-pro-glu-leu-leu-gly-gly-
arg-met-lys-gln-leu-glu-asp-lys-val-glu-glu-leu-leu-ser-lys-asn-tyr-his-leu-glu-asn-glu-val-ala-arg-leu-lys-lys-
leu-val-gly-glu-arg (SEQ. ID. NO: 56) . The resultant plasmid is named pXCA740.
The variable domains of the scFvs were amplified and restriction sites added for subcloning into the
vectors described above by the PCR technique. Specific oligonucleotides were designed for each V L or V H
35 region as shown below.
12B5, 12D5, and 10D10 Light chain variable domains:
5 primer
GCT TCT GCG GCC ACA CAG GCC TAC GCT GAC ATC GTG ATG ACC C (SEQ. ID. NO: 57)
3 primer
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ATG ATG ATG TGC CAC GGT CCG TTT GAT CTC CAG TTC GGT C (SEQ. ID. NO: 58)
12E10 Light chain variable domain:
5 primer
GCT TCT GCG GCC ACA CAG GCC TAC GCT TCC TAT GTG CTG ACT C (SEQ. ID. NO: 59)
3 primer
CCT TCT CTC TTT AGG TTG GCC AAG GAC GGT CAG CTT GGT C (SEQ. ID. NO: 60)
10F6 Light chain variable domain
5 primer
GCT TCT GCG GCC ACA CAG GCC TAC GCT CAG TCT GTG CTG ACT C (SEQ. ID. NO: 61)
3 primer
CCT TCT CTC TTT AGG TTG GCC AAG GAC GGT CAG CTT GGT C (SEQ. ID. NO: 60)
12B5 Heavy chain variable domain
5 primer
CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG GTG CAG (SEQ. ID. NO: 62)
3 primer
GTA AAT GTA TGG GCC CTT GGT GGA GGA GGC ACT CGA GAC GGT GAC (SEQ. ID. NO:
63)
I2D5 Heavy chain variable domain
5 primer
CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG GTG GAG (SEQ. ID. NO: 64)
3 primer
GTA AAT GTA TGG GCC CTT GGT GGA GGA GGC ACT CGA GAC GGT GAC (SEQ. ID. NO:
63)
10D10 Heavy chain variable domain
5 primer
CAT TCT ACA AAC GCG TAC GCT GAC GTG CAG CTG GTG CAG (SEQ. ID. NO: 65)
3 primer
GTA AAT GTA TGG GCC CTT GGT GGC GGC TGA GGA GAC GGT GAC (SEQ. ID. NO: 66)
12E10 Heavy chain variable domain
5 primer
CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG CAG CAG (SEQ. ID. NO: 67)
3 primer
GTA AAT GTA TGG GCC CTT GGT GGA GGA GGC ACT CGA GAC GGT GAC (SEQ. ID. NO:
63)
1 0F6 Heavy chain variable domain
5 primer
CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG CAG GAG (SEQ. ID. NO: 68)
3 primer
GTA AAT GTA TGG GCC CTT GGT GGA GGC TGA AGA GAC GGT AAC (SEQ. ID. NO: 69)
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PCR reactions were carried out using 100 ng of plasmid DNA containing the scFv, 0.5 uM of the
appropriate 5 and 3 primer, 200 uM each dNTP, 10 mM KC1, 6 mM(NH 4 ) 2 S0 4 , 20 mM TRIS-HCI, pH 8.0, 2
mM MgCl 2 , 1% Triton X-100, lOOuM BSA and 2.5 units of Pfu DNA polymerase (Stratagene) . Amplification
was for 30 cycles of: 30 sec at 95 °C, 30 sec at 55°C, 30 sec at 72°C. After digestion with the appropriate
5 restriction enzymes, the reaction products were separated by agarose gel electrophoresis and the approximately
350 bp band was isolated using a Gene Clean II kit (BIO 101, Vista, CA). The fragments for the light chain
variable regions were ligated into the vectors previously digested with Sfi I and Rsr II for the kappa isotypes, or
Sfi I and Msc I for the lambda isotypes, and transformed into E. coli DH5a. Desired recombinants were
identified using restriction enzyme analysis and sequenced to confirm the presence of the desired fragments.
10 The heavy chain variable domains were then cloned similarly into the plasmids containing the light chains using
the restriction enzymes Mlu I and Apa I, and the final constructions were again checked by DNA sequencing.
(k) Construction of scFv with gD tags.
For increased and regulated expression in high density fermentation tanks, the Sfi I to Not 1 fragments
15 of the scFv forms of pl2B5, pl2D5, plOF6, and p!2E10 were subcloned into a derivative of pAK.19 containing
the phoA promoter and stll signal sequence rather than the lacZ promoter and hybrid signal sequence of the
original library. For ease of purification, a DNA fragment coding for 12 amino acids (met-ala-asp-pro-asn-arg-
phe-arg-gly-lys-asp-leu) (SEQ. ID. NO: 70) derived from herpes simplex virus type 1 glycoprotein D (Lasky
and Dowbenko DNA (N.Y.) 3:23-29 (1984.)) was synthesized and inserted at the 3 end of the V L domain in
20 place of the (his) 6 and c-myc epitope originally present in the C.A.T. library clones.
(1) Expression in E. coli
Plasmids containing genes for scFv-gD, Fab' or Fab'2 molecules were expressed in E. coli strain 33B6
(W3110 DfhuA pho ADE1 5 deoCl //vG2096(val R ) cfegP4I(DPstI-ICan R ) D(argF-/ac) 1 69 IN(rr«D-/-r«E) 1 )
grown for approximately 40 hr at 30°C in an aerated 10-liter fermentor as described previously (Carter et al
25 Bio/Technology 10:163-167(1992.)).
Example 3
Cloning and expression of full length human antibody derivatives of 12B5. 12D5. and 12E10.
For expression of full length antibodies in mammalian cells, the heavy chain variable domains were
subcloned from the Fab constructs into a derivative of expression vector pRK (Suva et al.. Science 237:893-
30 896 (1987)) which contains the human IgGl CHI, CH2, and CH3 domains and a human antibody signal
sequence (Carter et al., Proc. Natl. Acad. Sci. USA. 89:4285-4289 (1992)). The light chain was cloned into a
separate pRK plasmid. The light and heavy chain expression vectors were cotransfected into adenovirus-
transformed human embryonic kidney cell line 293 by a high-efficiency procedure (Gorman et al, DNA Protein
Eng. Technol. 2:3-10 (1990)). Harvested conditioned media was shown to contain anti-mpl antibody by
35 ELISA.
For production of a more stable cell line and high-level antibody production, the light and heavy
chains were moved into the SVI.DI expression vector previously described (Lucas et al., Nucleic Acids Res. 24:
1774-1779 (1996). This vector contains the mouse DHFR cDNA in the intron of the expression vector pRK
and allows for amplification of expression by selection in methotrexate The light chain is cloned into the same
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plasm id with expression driven by a second SV40 promoter/ enhancer. The plasmid was linearized and
transfected into CHO cells using lipofectamine (Gibco-BRL) following manufacturer's Instructions. Seven to
ten days after transfer to selective medium, clones were isolated into 96 well plates for later study, or pooled
and expanded for culture in roller bottles.
5 Conditioned media for purification of the antibodies was generated in roller bottles. Cells were seeded
into the roller bottles at an initial cell density of 2 x 107 cells in 200 ml rich medium (DMEM: Ham's F12 (1:1)
supplemented with 5% fetal bovine serum. At approximately 80% confluency, the media was replaced with
serum-free PS-24 production medium supplemented with insulin (10 ug/ml), transferrin (10 ug/ml), trace
elements and lipid alcohol. Conditioned media was harvested after 1 0 days.
10 Example 4
Purification Of Agonist Antibodies
(a) Purification of scFv with gD tag
Frozen cell paste was resuspended at 1 gm/ml TE (25 mM TRIS, 1 mM EDTA, pH 7.4) and gently
agitated 1 8 hr on ice. Cell debris was removed by centrifugation at 10,000 x g for 30 min. The supernatant was
15 loaded onto an affinity column (2.5 x 9.0 cm) consisting of an anti-gD monoclonal antibody 5B6 (Paborsky, L.
R. et al., Protein Eng. 3: 547-553 (1990)) coupled to CNBr SEPHAROSE which had been equilibrated with
PBS. The column was washed 18 hr with PBS, and then washed with PBS containing 1 M NaCl until the
absorbance of the column effluent was equivalent to baseline. All steps were done at 4°C at a linear flow rate of
25 cm/hr. Elution was performed with 0.1 M acetic acid, 0.5 M NaCl, pH 2.9. Column fractions were
20 monitored by absorbance at 280 nm and peak fractions pooled, neutralized with 1.0 M TRIS, pH 8.0, dialyzed
against PBS, and sterile filtered. The resultant protein preparations were analyzed by non-reducing SDS-
PAGE.
(b) Purification of Fab' molecules
For purification of Fab' molecules, 5 g of frozen cell paste was resuspended in 5 ml of TE (25 mM
25 TRIS, 1 mM EDTA, pH 7.4) and gently stirred 18 hr on ice. The pH of the shockate was adjusted to 5.6 with 2
M HC1 and the precipitate and cell debris removed by centrifugation at 10,000 x g for 30 min. The supernatant
was loaded onto a 1 ml BAKERBOND ABx column (0.5 x 5.0 cm) (J. T. Baker, Phillipsburg, NJ) pre-
equilibrated with 20 mM MES, pH 5.5. After washing with 20 mM MES to baseline, the Fab' was eluted using
a 10 ml linear gradient from 0 to 100% of 20 mM NaOAc, 0.5 M (NH 4 ) 2 S0 4 , pH 7.2, with a flow rate of 153
30 cm/hr. Fractions containing Fab' were pooled, and buffer exchanged into PBS.
(c) Purification of Fab'2 molecules
Frozen cell paste (100 gm) was thawed into 10 volumes of 25 mM TRIS, 5 mM EDTA, 1 mM NaN3,
pH 7.4 and disrupted by three passages through a microfluidizer (TECH-MAR). PMSF was added to 1 mM and
the cell debris removed by centrifugation at 10,000 x g for 30 min. The supernatant was filtered sequentially
35 through a 0.45um, and a 0.2 urn SUPORCAP filter (Gelman), and loaded onto a 50 ml SEPHAROSE-fast-flow
Protein-G column (Pharmacia) pre-equilibrated with PBS. After washing to baseline with PBS, Fab'2 was
eluted with 0.1 M glycine ethyl ester, pH 2.3, into tubes with contained 1/10 volume of 1 M TRIS, pH 8.0.
Fractions containing Fab'2 were pooled and concentrated by Ultrasette with a 30 kilodalton molecular weight
cut off, and buffer exchanged into 20 mM NaOAc, 0.01% octylglucoside, pH 5.5. This material was loaded
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onto a 30 ml S-SEPHAROSE column (Pharmacia) pre-equilibrated with 20 mM NaOAc, washed to baseline
with 20 mM NaOAc, pH 5.5, and eluted with a linear gradient of 0-1 M NaCl in 25mM NaOAc over 1 0 column
volumes. Fractions containing Fab'2 were pooled and buffer exchanged to PBS.
(dl Purification of full length antibodies from transfected CHO cell supernatants .
5 Conditioned medium harvested from roller bottles was loaded onto a 5 ml Protein-A SEPHAROSE
column (1.0 x 5.0 cm) pre-equilibrated with PBS, washed with PBS, and then washed to baseline with PBS
containing 1 M NaCl. Antibody was eluted with 0.1 M HOAc, 0.5 N NaCl, pH 2.9, neutralized with 1 M TRIS,
and buffer exchanged to PBS.
A summary of agonist antibody activities for several antibodies and fragments thereof is shown in
10 Table 4 below.
Table 4
Summary of Mpl Agonist Antibody Activities
Antibody
Hu3 Proliferation
(ED50)
K.1RA
(ED50)
Hu3 Binding
(IC50)
Mpl/ I PO ELISiA
(IC50)
Platelets
(IC50)
MK. Assay
12B5
scFv
20 pM
1 nM
10 nM
17nM
100 nM
+ +
Fab
'>luM
3 nM
900 nM
none
>luM
Fab'2
5 pM
1 nM
5 nM
1 nM
300 nM
+
IgG
30 pM
400 pM
10 nM
152 pM
300 nM
12E10
scFv
5 pM
60 pM
5nM
1.6 nM
5nM
Fab
>luM
>luM
500 nM
180 nM
>luM
Fab'2
>luM
160 pM
10 nM
640 nM
500 nM
IgG
>luM
480 pM
50 nM
450 pM
500 nM
12155
scFv
1.2 nM
280 pM
10 nM
24 nM
>luM
Fab
>luM
4nM
500 nM
luM
>luM
Fab'2
4.8 pM
600 pM
4 nM
1 nM
100 nM
+
IgG
>luM
3 nM
10 nM
450 pM
500 nM
Example 5
15 In another embodiment, the invention provides a method of selecting an antibody which binds to and
dimerizes a receptor protein. In this method, a library of antibodies is panned using a receptor protein having
two protein subunits as the binding target. The library is panned as described above for mpl agonist antibodies.
Preferably, the antibodies are human and more preferably monoclonal. The library is conveniently a library of
single chain antibodies, preferably displayed on the surface of phage. The display of proteins, including
20 antibodies, on the surface of phage is well known in the art as discussed above and these known methods may
be used in this invention. Antibody libraries are also commercially available, for example, from Cambridge
Antibody Technologies (CAT), Cambridge, UK. Preferably, the antibody selected by the method of the
invention activates the receptor by dimerizing the receptor and thereby achieves an effector result similar to the
effector result generated when the natural endogenous ligand for the receptor binds the receptor.
25 The method of the invention can be used to find agonist antibodies to any receptor having two
components which is known and /or can be cloned. It is not necessary to know the primary, secondary or
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tertiary structure of the receptor protein, although this information is useful for cloning, etc., since the method
of the invention allows selection of antibodies which will bind any displayed receptor which is activated by
dimerization. Many known receptor proteins are activated by dimerization and any of these known receptors
may be used in the invention. Suitable receptors include tyrosine kinase receptors and hematopoietic receptors
5 that lack kinase activity.
Activation of a receptor such as a tyrosine kinase receptor by a scFv is an unexpected result. Current
understanding of receptor activation argues that for many classes of receptors, including tyrosine kinase
receptors and hematopoietic cytokine receptors that lack intrinsic tyrosine kinase activity (but associate with
intracellular kinases), it is a dimerization event mediated by a ligand that is the key event in receptor activation.
10 This view is supported by crystal structures of receptor ligand complexes as well as the demonstrated agonist
ability of certain monoclonal antibodies (but not the Fab' fragments of these antibodies). A single chain
antibody would not, therefore, be expected to be able to cause receptor dimerization and activation.
MuSK is a recently identified tyrosine kinase localized to the postsynaptic surface of the
neuromuscular junction. (Valenzuela et. ai. 1995. Neuron 15 573-584.) Mice made deficient in MuSK fail
15 to form neuromuscular junctions (Dechiara et. al.. 1996. Cell 85 501-512.), a phenotype highly similar to that
observed in mice lacking the nerve derived signaling molecule agrin (Gautam et. ai, 1996, Cell 85 525-535).
The likely involvement of MuSK in agrin signaling is strengthened by the observations that agrin induces the
rapid tyrosine phosphorylation of MuSK and that labeled agrin can be chemically crosslinked to MuSK (Glass
et. al., 1996, Cell 85 513-523.) .
20 Formation of the neuromuscular junction is achieved through a process that includes the differentiation
of membrane on the muscle fiber proximal to the neuron terminus and changes in gene expression within the
nuclei proximal to this junction (reviewed by Bowe et. ai. 1995, Annu-Rev-Neurosci. 18 443-462 and Kleiman
et. ai, 1996, Cell 85 461-464.). A striking feature of this complex process is the redistribution and
concentration of AChRs within the myotube membrane. Agrin is able to the induce this clustering of AChRs as
25 well as changes in the extracellular matrix and cytoskeletal components of the synaptic apparatus (Bowe et. ai,
supra; Godfrey et. a/.,1984, J. Cell Biol. 99 615-627; Nitkin et. ai, 1987, J. Cell Biol. 105 2471-2478).
Agrin is a secreted protein with a core molecular weight of -200 kDa that contains several copies of EGF
repeats, laminin-like globular domains and sequences that resemble protease inhibitors. It is released by motor
neuron terminals and maintained within the basil lamina of the synaptic cleft. While agrin apparently does not
30 to bind MuSK with high affinity (Glass et. al., supra), it has been reported to interact with other molecules
present at the neuromuscular junction, most notably alpha-dystroglycan (O'Toole et. al., 1996,. Natl. Acad Sci.
USA 93 7369-7374) thereby complicating the analysis of MuSK's role in the signaling events initiated by agrin.
Antigen specific scFv, identified by panning a diverse library of scFv expressed, for example, on M13
phage provide a source of molecules capable of mediating specific therapeutic activities, and offer a rapid new
35 approach to study the function of novel or recently identified molecules such as MuSK. scFv are identified
below that mediate receptor activation and that direct MuSK activation induces changes in AChR distribution
and tyrosine phosphorylation similar to that observed with agrin.
The induction of AChR clustering and tyrosine phosphorylation by scFv antibodies provides direct
evidence to support conclusions drawn from studies of knockout mice deficient in MuSK indicating this
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recently discovered tyrosine kinase acts to induce key events in the formation of the neuromuscular junction.
As a potential signal transducer of agrin, it is noteworthy that MuSK does not display high affinity binding to
agrin, leading to speculation that there must be an additional agrin binding component(s) involved in mediating
the agrin signal . The molecular nature of this component is unknown. It is interesting that it is possible to
5 induce the receptor clustering, the hallmark activity of agrin, with an agent directed specifically to MuSK.
The marked upregulation of MuSK expression in muscle following denervation or muscle
immobilization as well as the chromosomal localization of MuSK within a region associated with fukiyama
muscular dystrophy point to an important role for this molecule in regulation of the neuromuscular junction
(Valenzuela et.al., supra) and indicates the possibility that therapeutic benefit is possible through the controlled
10 regulation of MuSK activity. As agrin is expressed not only at the neuromuscular junction, but in a wide
variety of peripheral and central neurons (Bowe et. ai, supra; Rupp et. ai, 1991, Neuron 6 81 1-823; Tsim et.
al.., 1992, Neuron 8 677-689) it may not be an optimal candidate molecule through which to manipulate MuSK
function as exogenously introduced agrin derivatives might elicit consequences not restricted to the
neuromuscular junction. Thus, in comparison, the ability to obtain direct activation of MuSK through scFv
15 offers an attractive alternative. Each of the scFv that were tested displayed affinity for MuSK in the nM range
demonstrating the utility of phage displayed scFv libraries as a rich source of high affinity and highly specific
molecules.
The antibodies of the invention are, therefore, useful in assaying the upregulation of MuSK receptors
in sample tissues to determine the degree of neuromuscular damage associated with this upregulation. The
20 antibodies are also useful for activating the MuSK receptor and inducing AChR clustering at neuromuscular
junctions as a direct result of the agonist properties of these antibodies. Administration of the antibodies to a
person suffering from denervation or muscle immobilization, e.g. muscular dystrophy, provides a method of
improving the function of the neuromuscular junctions in these people.
To prepare scFv having agonist activity, antibodies were selected which induce a proliferative
25 response in a factor dependent cell line through a chimeric MuSK-Mpl receptor comprised of the extracellular
domain of MuSK and the intracellular domain of the hematopoietic cytokine receptor c-Mpl (the receptor for
thrombopoietin, TPO). Activation of c-Mpl is believed to require homodimerization, as is the case for the
growth hormone receptor, the erythropoietin receptor and other related receptors of this class (Carter el.
ai, 1996, Annu-Rev-Physiol 58 187-207; Gumey et. ai, 1995, Proc .Natl. Acad. Sci. USA 92 5292-5296).
30 Ba/F3 cells expressing MuSK-Mpl were starved of IL-3 and exposed to a range of concentrations of each scFv
expressed as soluble protein. Surprisingly, 4 of the 21 scFv were able to induce a robust proliferative response
in the MuSK-Mpl expressing cells (Fig. 1 1 ). This activity was observed at nM concentrations of scFv. The
scFv were without effect on the parental, untransfected Ba/F3. Agonist activity was also present among those
IgG that were derived from agonist scFv but was not noted among IgG derived from non-agonist scFv. Soluble
35 agrin c-terminal domain (c-agrin) was without effect supporting previous observations that agrin does not bind
MuSK directly. The c-terminal domain of agrin is known to contain the AChR clustering activity of agrin and
is essential for neuromuscular junction formation (Ruegg et ai., 1992, Neuron 8 691-699; Tsim et. al, supra).
The EC50 for the ability to induce proliferation was 5 nM for the most active agonist clone when expressed as
either scFv or IgG. The affinity of these scFv and IgG for MuSK was determined using BIAcore^M analysis.
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The agonist scFv and several non-agonist scFv each displayed affinity for MuSK within the range of 5-25 nM.
In contrast, the affinities of the IgG for MuSK were 10-30 pM . See Table 5 below.
Table 5
clone #
Agonist
k d .
Affinity
musk #2-scfrv
+
3.34 x
8.78 x 10*
3.8 nM
musk #3-scFv
2.39 x
10-3
1.05 x 10 5
23 nM
musk #4-scFv
+
1.57 x
10-3
1.84 x 10 5
8.5 nM
musk #5-scFv
2.49 x
10-3
5.29 x 10 5
4.7 nM
musk #6-scFv
4.95 x
10-3
1.05 x 10 5
4.7 nM
musk #13-scFv
+
2.32 x
10-3
4.53 x 10 5
5.1 nM
musk #22-scFv
+
6.09 x
10-3
1.27 xlO 5
4.8 nM
musk#13-IgG
+
1.01 x
10-5
8.05 x 10 5
12.5 pM
musk #22-IgG
+
4.86 x
10-5
1.65 x 10 6
29.5 pM
To probe this agonist
activity
further, scFv
were
examined for the
ability to induce
phosphorylation of full length MuSK tyrosine kinase. The murine myoblastic cell line C2CI2 was cultured
under conditions that promote myotube differentiation and subsequently exposed to scFv, IgG or c-agrin. In
correspondence with previous data (Glass et. at, supra), c-agrin was able to induce MuSK tyrosine
phosphorylation. The agonist scFv and IgG were also found to rapidly induce tyrosine phosphorylation of
10 MuSK as determined by western blot analysis with anti-phosphotyrosine antibody whereas other scFv and non-
agonist anti-MuSK IgG were without effect.
The ability of the scFv MuSK agonists to induce AChR clustering in cultured C2C12 myotubes was
examined. Following stimulation, the cells were fixed and the distribution of cell surface AChR was revealed
with rhodamine labeled bungarotoxin. In undifferentiated myoblasts, AChR were dispersed and unfocused in
15 the presence of c-agrin, scFv, or IgG. In contrast, upon myotube differentiation, c-agrin and agonist scFv and
IgG induced marked aggregation of AChR into large and intensely stained clusters. Non-agonist scFv and non-
agonist IgG directed against MuSK or an irrelevant antigen were without effect. An additional consequence of
agrin action, tyrosine phosphorylation of subunits of the AChR was also examined utilizing an antisera that
recognizes the and chains of the receptor. Tyrosine phosphorylation levels of both the and chains were
20 markedly induced by c-agrin as well as the agonist scFv and agonist IgG but were unaffected by control scFv
and IgG.
Variants of the MuSK agonist antibodies of the invention may be prepared as described above for
thrombopoietic antibodies.
Construction of expression vectors . Coding sequence for murine MuSK was obtained by PCR
25 amplification. MuSK-Fc was prepared by fusion of the extracellular domain of MuSK (a.a. 1-492) in frame
with the Fc region of human IgG 1 in the eukaryotic expression vector pRK5tkNEO. MuSK-Fc was transiently
expressed in 293 cells and purified over a protein G column. A chimeric receptor, MuSK-MpI, comprised of
the extracellular domain of MuSK (amino acids 1-492) and the transmembrane and intracellular domain of the
human c-Mpl receptor (amino acids 491-635) was prepared by sequential PCR and cloned into pRK5tkNEO.
30 Stable cell lines expressing the chimeric receptor were obtained by electroporation (5 million cells, 250 volts,
960 u,F) of linearized vector (20 ug) into Ba/F3 cells followed by selection for neomycin resistance with 2
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mg/ml G4I8. Full length MuSK in pRK5tkNEO was transfected into 293 cells and stable transformants were
obtained following two weeks of G418 selection (400 ug/ml). The sequence of the DNA constructs were
confirmed by DNA sequencing. Expression of MuSK was assessed by flow cytometry analysis as described
below. Ba/F3 cells were maintained in RPMI 1640 media supplemented with 10% fetal calf serum and 5%
5 conditioned media from WEHI-3B cells as source of IL-3. C-agrin (amino acids 1 137-1949 of the rat agrin (Ag
+8 active splice form (Fems et al, 1993, Neuron 1 1 491-502.)) was expressed by transient transfection from
293 cells in serum free media with an expression vector, pRK-gD-c-Agrin, as a fusion protein with the gD
signal sequence and epitope tag and a genenase cleavage site (MGGAAARLGAVILFVV
IVGLHGVRGKYALADASLKMADPNRFRGKDLPVLDQLLEGGAAHYALLPG) (SEQ ID NO. 71) fused to
1 0 the N-terminus.
Isolation of scFv and IgG MuSK-Fc immunoadhesin was coated on Maxisorp tubes (Nunc) at 10
ug/ml. A library of human scFv (Cambridge Antibody Technology, England) was panned through two rounds
of enrichment essentially as described (Griffiths et al, 199, EMBO-J 12 725-734). The specificity of individual
clones was assessed first by elisa (Griffiths et al, supra) using MuSK-Fc and a control immunoadhesin (CD4-
15 Fc). Positive clones were screened by PCR and "fingerprinted" by BstNl digestion (Clackson et al, 1991,
Nature 352 642-648.). Examples of clones with unique patterns were sequenced and subjected to FACS
analysis with cells expressing or not expressing MuSK. For FACS analysis, cells (10 5 ) were incubated for 60
minutes at 4 C in 200 ul 2% FBS/PBS (fetal bovine serum/phosphate buffered saline) with 10 10 phage that
were first blocked by incubation in 30 ul 10% FBS/PBS. Cells were then washed with 2% FBS/PBS, stained
20 with anti-M13 antibody (Pharmacia, Piscataway NJ) and R-phycoerytherin-conjugated donkey anti-sheep
antibody (Jackson Immunoresearch, West Grove PA), and analyzed by FACS analysis. ScFv were expressed in
bacteria as epitope tagged proteins containing a c-myc tag sequence recognized by monoclonal antibody 9E10
(Griffiths et al, supra) and a polyhistidine tail (hisg) and were purified over Ni-NTA column with imidazole
elution as recommended by manufacturer (Qiagen). For expression of clones as IgG the sequences encoding
25 the Vh and Vl regions of the scFv were introduced by PCR into mammalian expression vector plgG-kappa
which was designed to enable the expression of fully human light and heavy chains of kappa type IgG.
Expression vectors for the individual clones were transfected into CHO cells and IgG were harvested from
conditioned serum free media and purified over a protein A column.
Proliferation assays . Cells were cultured in the absence of IL-3 for twenty-two hours (in RPMI
30 supplemented with 10% FBS). Cells were then washed twice with RPMI and plated in 96 well dishes at 50,000
cells per well in 0.2 ml of 7.5% FBS RPMI supplemented with the indicated concentrations of scFv or IgG.
Each concentration was tested in duplicate. After an incubation of sixteen hours, 1 uCi of [^HJ-thymidine was
added per well and incubation was continued for an additional six hours. Incorporation of [-^-thymidine was
measured with a Top Count Counter (Packard Instruments, CA).
35 AChR clustering assay . C2C12 were maintained in 10% FBS in high glucose DMEM at subfluency.
For AChR clustering assays C2C12 were seeded on glass slides coated with fibronectin and poly-lysine and
myotube differentiation was induced by 48 hour incubation in 2% horse serum high glucose DMEM. scFv or c-
agrin were added to the culture medium and incubated overnight (16 hours). Cells were then washed with PBS
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GRIFFITH HACK
11010
and fixed in 4Vi paraformaldehyde. Rhodaminc conjugated bungarotexin (Molecular Probes, Eugene Oft) was
used to reveal the localization of AChRs as described (Ferns et al, supra).
i
Binding affinity analysis . Protein interaction analysis using BIAcoreTM instruments was performed as
described (Mark el al, i99€, J. Biol. Chcm. 271 9785-9789). Briefly, research grade CMS sensor chips were
5 activated by injection of 20 ul of 1:1 N-Ethyl-W-(3-dimcthylamuiopropyl)carboiimide hydrochloride and N-
hydroxysuccinimidc at 5 ul /rain flow rate. 20 ul of MuSK-Fc at 20 ugAain in 10 raM sodium acetate, pH 5.0
was injected over the sensor chip, followed by 30 ul of ethanolamine. scFv or IgG were purified and
concentrations determined by Pcarce BCA kit Thirty ul protein samples in PBS with 0.05% Tween 20 were
injected at a flow rate of 10 ul min by the K inject method. Proteins were allowed to dissociate for 20 tnin in a
10 flow of PBS with 0.05% Tween 20. Sctuorgrams were analyzed with BlAevaluarion 2.1 software from
Pharmacia Biosensor AB. Apparent dissociation rate constants (Jty) and association rate constants (k#) were
obtained by evaluating the sensorgram with A + B- AB type I fitting. Equilibrium dissociation constant
was calculated as kdfka.
Immunoprecipitation and Western Blot . Analysis. C2C12 were maintained in 10% FBS high glucose
IS DMEM and induced to differentiate by 72 hour incubation in 2% horse scrum. Cells were then stimulated by
addition of c-agrin, scFv or IgG for the time indicated in the figures. ScFv and IgG were used at SO nM. The e-
agrin containing conditioned media was used at level that provided maximal tyrosine phosphorylation. Cell
extracts were prepared as described (Gumey et al. supra) . Extracts were incubated for CO minutes at 4°C with
30 ul agarose conjugated antiphosphotyrosine monoclonal antibody 4G10 (UBI ino. Lake Placid NY) or 1 jig
20 of ano-MuSK. IgG #13 followed by 30 ul protein A sepharose beads. Western blot analysis with
antiphosphotyrosine antibody 4G10 or ami AChR antibody (Affinity Bioreagents, Golden CO) was performed
as recommended by the manufacturer and revealed with HRP conjugated secondary antibody and CCL
(Amersham).
MuSK scFv arc readily observed as doners when resolved by oondenaturing gel electrophoresis.
25 Additionally, the abundance of dimeric species may be significantly altered in the local context of scFv bound
to receptor on the cell surface. Alternatively, screening an jeFv phage library with a divalent antigen, in this
case MuSK-Fc, allows direct selection of scFv that bind to and facilitate the formation Of a receptor dimer.
While the invention has necessarily been described in conjunction with preferred embodiments and
specific working examples, one of ordinary skill, after reading the foregoing specification, will be able to effect
30 various changes, substitutions of equivalents, and alterations to the subject matter set forth herein, without
departing from the spirit and scope thereof. Hence, the invention can be practiced in ways other than those
specifically described herein. It is therefore intended that the protection granted by letters patent hereon be
limited only by the appended claims and equivalents thereof.
All references cited herein are hereby expressly incorporated by reference.
35 Deposit of Material
The following materials have been deposited with the American Type Culture Collection,
10801 University Boulevard, Manassas, Virginia, 201 10-2209 USA (ATCC):
Material ATCC Deo. No. Deposit Date
pMpl.l2BS.scFv.his 203125 August 18, 1998
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29/10 '02 TUE 11:44 FAX 61 7 3221 1245
GRIFFITH HACK
Hon
pMpU2D5.scFv.his 203121 August 18, 1998
pMpU2E10.scFv.his 203120 August 1 8. 1998
pMpL10D10.SCFv.his 203124 August 18, 1998
pMpLlOFd.scFv.his 203122 August 1 8. 1998
5 pMpL5E5.scFY.his 2031 S5 August 18, 1998
This deposit Was made under the provisions of the Budapest Treaty on the International Recognition of
the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest
Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the dale of deposit. The
deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement
10 between Genentech, Inc. and ATCC. which assures permanent and unrestricted availability of the progeny of
the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the
public of any US. or foreign patent application, whichever comes fust, and assures availability of the progeny
to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35
USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR § M4 with particular reference to
IS 886 OO 638).
The assignee of the present application has agreed that if a culture of the materials On deposit should
die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on
notification with another ef the same. Availability of the deposited material is not to be construed as a license
to practice the invention in contravention of the rights granted under the authority of any government in
20 accordance with its patent laws.
The foregoing written specification is. considered to be sufficient to enable one skilled in the art to
practice the invention. The present invention Is not to be limited in scope by the construct deposited, since the
deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs
that are functionally equivalent are within the scope of this invention. The deposit of material herein docs not
25 constitute an admission that the written description herein contained is inadequate to enable the practice of any
aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the
claims to the Specific illustrations that it represents. Indeed, various modifications of the invention in addition
to those shown and described herein will become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
30
-57-
EDITORIAL NOTE
PATENT APPLICATION 88312/98
The following sequence listing form
part of the specification.
The claims resume at page 58
WO 99/10494
PCT/US98/17364
Sequence Listing
<110> Genentech, Inc.
<120> Agonist Antibodies
10 <130> P0979R1
<150> US 60/056,736
<151> 1997-08-22
15 <160> 71
<210> 1
<211> 15
<212> DNA
20 <213> human
<400> 1
acc tct tgg ate ggc 15
Thr Ser Trp He Gly
25 15
<210> 2
<211> 5
<212> PRT
30 <213> human
<400> 2
Thr Ser Trp He Gly
1 5
35
<210> 3
<211> 66
<212> DNA
< 2 1 3 > human
<400> 3
ate atg tat cct ggg aac tct gat acc aga cac aac 36
He Met Tyr Pro Gly Asn Ser Asp Thr Arg His Asn
15 10
ccg tec ttc gaa gac cag gtc acc atg tea 66
Pro Ser Phe Glu Asp Gin Val Thr Met Ser
15 20 22
50 <210> 4
<211> 22
<212> PRT
< 2 1 3 > human
55 <400> 4
lie Met Tyr Pro Gly Asn Ser Asp Thr Arg His Asn Pro Ser Phe
15 10 15
Glu Asp Gin Val Thr Met Ser
60 20 22
40
45
<210> 5
Sequence Listing
- 1 -
WO 99/10494
PCT/US98/17364
<211> 30
<212> DNA
<213> human
5 <400> 5
get ggg gtc gcg ggc ggt get ttt gat etc 30
Ala Gly Val Ala Gly Gly Ala Phe Asp Leu
15 10
10 <210> 6
<211> 10
<212> PRT
<213> human
15 <400> 6
Ala Gly Val Ala Gly Gly Ala Phe Asp Leu
15 10
<210> 7
20 <211> 42
<212> DNA
<213> human
<400> 7
25 act gga acc age agt ggc gtt ggt ggt tat aac tat 36
Thr Gly Thr Ser Ser Gly Val Gly Gly Tyr Asn Tyr
1 5 10
gtc tec 42
30 Val Ser
14
<210> 8
<211> 14
35 <212> PRT
<213> human
<400> 8
Thr Gly Thr Ser Ser Gly Val Gly Gly Tyr Asn Tyr Val Ser
40 1 5 10 14
<210> 9
<211> 21
<212> DNA
45 <213> human
<400> 9
ggt aac age aat egg ccc tea 21
Gly Asn Ser Asn Arg Pro Ser
50 1 5 7
<210> 10
<211> 7
<212> PRT
55 <213> human
<400> 10
Gly Asn Ser Asn Arg Pro Ser
1 5 7
60
<210> 11
<211> 30
Sequence Listing
WO 99/10494
<212> DNA
<213> human
<400> 11
5 age aca tat gca ccc ccc ggt att att atg 30
Ser Thr Tyr Ala Pro Pro Gly lie lie Met
1 5 10
<210> 12
10 <211> 10
<212> PRT
<213> human
<400> 12
15 Ser Thr Tyr Ala Pro Pro Gly lie lie Met
15 10
<210> 13
<211> 15
20 <212> DNA
<213> human
<400> 13
gac tac tac atg age 15
25 Asp Tyr Tyr Met Ser
1 5
<210> 14
<211> 5
30 <212> PRT
<213> human
PCT/US98/17364
35
<400> 14
Asp Tyr Tyr Met Ser
1 5
40
<210> 15
<211> 66
<212> DNA
<213> human
45
<400> 15
tac att agt agt agt ggt agt ace ata tac tac gca 36
Tyr lie Ser Ser Ser Gly Ser Thr lie Tyr Tyr Ala
15 10
50
55
gac tct gtg aag ggc cga ttc acc ate tec 66
Asp Ser Val Lys Gly Arg Phe Thr lie Ser
15 20 22
<210> 16
<211> 22
<212> PRT
<213> human
<400> 16
Tyr lie Ser Ser Ser Gly Ser Thr lie Tyr Tyr Ala Asp Ser Val
1 5 10 15
60 Lys Gly Arg Phe Thr lie Ser
20 22
Sequence Listing
- 3 -
WO 99/10494
PCT/US98/17364
<210> 17
<211> 27
<212> DNA
<213> human
5
<400> 17
tgg agt ggt gag gat get ttt gat ate 27
Trp Ser Gly Glu Asp Ala Phe Asp lie
15 9
10
<210> 18
<211> 9
<212> PRT
<213> human
15
<400> 18
Trp Ser Gly Glu Asp Ala Phe Asp lie
1 5 9
20 <210> 19
<211> 33
<212> DNA
<213> human
25 <400> 19
egg gee agt gag ggt att tat cac tgg ttg gee 33
Arg Ala Ser Glu Gly He Tyr His Trp Leu Ala
1 5 10 11
30 <210> 20
<211> 11
<212> PRT
<213> human
35 <400> 20
Arg Ala Ser Glu Gly He Tyr His Trp Leu Ala
1 5 10 11
* <210> 21
40 <211> 21
<212> DNA
<213> human
<400> 21
45 aag gec tct agt tta gee agt 21
Lys Ala Ser Ser Leu Ala Ser
1 5 7
<210> 22
50 <211> 7
<212> PRT
<213> human
<400> 22
55 Lys Ala Ser Ser Leu Ala Ser
1 5 7
<210> 23
<211> 27
60 <212> DNA
<213> human
Sequence Listing
- 4 -
WO 99/10494
PCT/US98/17364
10
<400> 23
caa caa tat agt aat tat ccg etc act 27
Gin Gin Tyr Ser Asn Tyr Pro Leu Thr
1 5 9
<210> 24
<211> 9
<212> PRT
<213> human
<400> 24
Gin Gin Tyr Ser Asn Tyr Pro Leu Thr
1 5 9
15 <210> 25
<211> 15
<212> DNA
<213> human
20 <400> 25
acc tac ggc atg cac 15
Thr Tyr Gly Met His
1 5
25 <210> 26
<211> 5
<212> PRT
<213> human
30 <400> 26
Thr Tyr Gly Met His
1 5
<210> 27
35 <211> 66
<212> DNA
<213> human
<400> 27
40 ggt ata tec ttt gac gga aga agt gaa tac tat gca 36
Gly lie Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala
15 10
gac tec gtg aag ggc cga ttc acc ate tec 66
45 Asp Ser Val Lys Gly Arg Phe Thr lie Ser
15 ' 20 22
<210> 28
<211> 22
50 <212> PRT
<213> human
<400> 28
Gly lie Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala Asp Ser Val
55 1 5 10 15
Lys Gly Arg Phe Thr lie Ser
20 22
60 <210> 29
<211> 27
<212> DNA
Sequence Listing - 5 -
WO 99/10494
PCT/US98/17364
<213> human
<400> 29
gat agg ggg tec tac ggt atg gac gtc 27
5 Asp Arg Gly Ser Tyr Gly Met Asp Val
1 5 9
<210> 30
<211> 9
10 <212> PRT
<213> human
<400> 30
Asp Arg Gly Ser Tyr Gly Met Asp Val
15 1 5 9
<210> 31
<211> 66
<212> DNA
20 <213> human
<400> 31
ggt ata tec ttt gac gga aga agt gaa tac tat gca 36
Gly lie Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala
25 1 5 10
gac tec gtg cag ggc cga ttc acc ate tec 66
Asp Ser Val Gin Gly Arg Phe Thr lie Ser
15 20 22
30
35
<210> 32
<211> 22
<212> PRT
<213> human
<400> 32
Gly lie Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala Asp Ser Val
15 10 15
40 Gin Gly Arg Phe Thr lie Ser
20 22
<210> 33
<211> 24
45 <212> DNA
<213> human
<400> 33
gga gca cat tat ggt ttc gat ate 24
50 Gly Ala His Tyr Gly Phe Asp lie
1 5*8
<210> 34
<211> 8
55 <212> PRT
<213> human
<400> 34
Gly Ala His Tyr Gly Phe Asp lie
60 1 5 8
<210> 35
Sequence Listing - 6 -
WO 99/10494
PCT/US98/17364
<211> 33
<212> DNA
<213> human
5 <400> 35
egg gec age gag ggt att tat cac tgg ttg gec 33
Arg Ala Ser Glu Gly lie Tyr His Trp Leu Ala
1 5 10 11
10 <210> 36
<211> 15
<212> DNA
<213> human
15 <400> 36
age cat aac atg aac 15
Ser His Asn Met Asn
1 5
20 <210> 37
<211> 5
<212> PRT
<213> human
25 <400> 37
Ser His Asn Met Asn
1 5
<210> 38
30 <211> 66
<212> DNA
<213> human
<400> 38
35 tec att agt agt agt agt agt tac ata tac tac gca 36
Ser lie Ser Ser Ser Ser Ser Tyr lie Tyr Tyr Ala
1 5 10
gac tea gtg aag ggc cga ttc acc ate tec 66
40 Asp Ser Val Lys Gly Arg Phe Thr lie Ser
15 20 22
<210> 39
<211> 22
45 <212> PRT
<213> human
<400> 39
Ser lie Ser Ser Ser Ser Ser Tyr lie Tyr Tyr Ala Asp Ser Val
50 1 5 10 15
Lys Gly Arg Phe Thr lie Ser
20 22
55 <210> 40
<211> 27
<212> DNA
<213> human
60 <400> 40
gat cgc ggg agt acc ggt atg gac gtc 27
Asp Arg Gly Ser Thr Gly Met Asp Val
Sequence Listing - 7 -
WO 99/10494
PCT/US98/17364
<210> 41
<211> 9
<212> PRT
<213> human
10
<400> 41
Asp Arg Gly Ser Thr Gly Met Asp Val
15 9
15
<210> 42
<211> 15
<212> DNA
<213> human
20
<400> 42
agt tac tac tgg age
Ser Tyr Tyr Trp Ser
1 5
15
25
<210> 43
<211> 5
<212> PRT
<213> human
30
35
40
<400> 43
Ser Tyr Tyr Trp Ser
1 5
<210> 44
<211> 63
<212> DNA
<213> human
<400> 44
tat ate tat tac agt ggg age acc aac tac aac ccc 36
Tyr lie Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro
15 10
tec etc aag agt cga gtc acc ata tea
Ser Leu Lys Ser Arg Val Thr lie Ser
15 20 21
63
45 <210> 45
<211> 21
<212> PRT
<213> human
50 <400> 45
Tyr lie Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
15 10 15
Ser Arg Val Thr lie Ser
55 20 21
60
<210> 46
<211> 18
<212> DNA
<213> human
<400> 46
Sequence Listing
8
WO 99/10494
PCT/US98/17364
ggg agg tat ttt gac gtc 18
Gly Arg Tyr Phe Asp Val
1 5 6
5 <210> 47
<211> 6
<212> PRT
<213> human
10 <400> 47
Gly Arg Tyr Phe Asp Val
1 5 6
<210> 48
15 <211> 42
<212> DNA
<213> human
<400> 48
20 act gga acc age agt gac gtt ggt ggt tat aac tat 36
Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr
1 5 • ■ 1Q
25
gtc tec
Val Ser
14
42
<210> 49
<211> 14
30 <212> PRT
<213> human
35
<400> 49
Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser
1 5 10 14
40
<210> 50
<211> 21
<212> DNA
<213> human
45
<400> 50
gag ggc agt aag egg ccc tea 21
Glu Gly Ser Lys Arg Pro Ser
1 5 7
50
<210> 51
<211> 7
<212> PRT
<213> human
55
60
<400> 51
Glu Gly Ser Lys Arg Pro Ser
1 5 7
<210> 52
<211> 30
<212> DNA
<213> human
<400> 52
age tea tat aca acc agg age act cga gtt 30
Sequence Listing - 9 -
WO 99/10494
PCT/US98/17364
Ser Ser Tyr Thr Thr Arg Ser Thr Arg Val
1 5 10
<210> 53
5 <211> 10
<212> PRT
<213> human
<400> 53
10 Ser Ser Tyr Thr Thr Arg Ser Thr Arg Val
15 10
<210> 54
<211> 23
15 <212> DNA
<213> artificial sequence
<220>
<221> Sequence is completely synthesized
20 <222> 1-23
<400> 54
agcggataac aatttcacac agg 23
25 <210> 55
<211> 21
<212> DNA
<213> artificial sequence
30 <220>
<221> Sequence is completely synthesized
<222> 1-21
<400> 55
35 gtcgtctttc cagacggtag t 21
<210> 56
<211> 44
<212> PRT
40 <213> artificial sequence
<220>
<221> Sequence is completely synthesized
<222> 1-4 4
45
<400> 56
Cys Pro Pro Cys Ala Pro Glu Leu Leu Gly Gly Arg Met Lys Gin
1 5 10 15
50 Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr His Leu
20 25 30
Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly Glu Arg
35 " 40 44
55
60
<210> 57
<211> 43
<212> DNA
<213> artificial sequence
<220>
<221> Sequence is completely synthesized
Sequence Listing - 10 -
WO 99/10494
PCT/US98/17364
<222> 1-43
<400> 57
gcttctgcgg ccacacaggc ctacgctgac atcgtgatga ccc 43
5
<210> 58
<211> 40
<212> DNA
<213> artificial sequence
10
<220>
<221> Sequence is completely synthesized
<222> 1-40
15 <400> 58
atgatgatgt gccacggtcc gtttgatctc cagttcggtc 40
<210> 59
<211> 43
20 <212> DNA
<213> artificial sequence
<220>
<221> Sequence is completely synthesized
25 <222> 1-43
<400> 59
gcttctgcgg ccacacaggc ctacgcttcc tatgtgctga etc 43
30 <210> 60
<211> 40
<212> DNA
<213> artificial sequence
35 <220>
<221> Sequence is completely synthesized
<222> 1-40
<400> 60
40 ccttctctct ttaggttggc caaggaeggt cagcttggtc 40
<210> 61
<211> 43
<212> DNA
45 <213> artificial sequence
<220>
<221> Sequence is completely synthesized
<222> 1-43
50
<400> 61
gcttctgcgg ccacacaggc ctacgctcag tctgtgctga etc 43
<210> 62
55 <211> 39
<212> DNA
<213> artificial sequence
<220>
60 <221> Sequence is completely synthesized
<222> 1-39
Sequence Listing
-11-
WO 99/10494 PCT/US98/17364
<400> 62
cattctacaa acgcgtacgc tcaggtgcag ctggtgcag 39
<210> 63
5 <211> 45
<212> DNA
<213> artificial sequence
<220>
10 <221> Sequence is completely synthesized
<222> 1-45
<400> 63
gtaaatgtat gggcccttgg tggaggaggc actcgagacg gtgac 45
<210> 64
<211> 39
<212> DNA
<213> artificial sequence
<220>
<221> Sequence is completely synthesized
<222> 1-39
15
20
25 <400> 64
cattctacaa acgcgtacgc tcaggtgcag ctggtggag 39
<210> 65
<211> 39
30 <212> DNA
<213> artificial sequence
<220>
<221> Sequence is completely synthesized
35 <222> 1-39
<400> 65
cattctacaa acgcgtacgc tgacgtgcag ctggtgcag 39
40 <210> 66
<211> 42
<212> DNA
<213> artificial sequence
45 <220>
<221> Sequence is completely synthesized
<222> 1-42
<400> 66
50 gtaaatgtat gggcccttgg tggcggctga ggagacggtg ac 42
<210> 67
<211> 39
<212> DNA
55 <213> artificial sequence
<220>
<221> Sequence is completely synthesized
<222> 1-39
60
<400> 67
cattctacaa acgcgtacgc tcaggtgcag ctgcagcag 39
Sequence Listing - 1 2 -
WO 99/10494 PCT/US98/17364
<210> 68
<211> 39
<212> DNA
5 <213> artificial sequence
<220>
<221> Sequence is completely synthesized
<222> 1-39
10
25
30
60
<400> 68
cattctacaa acgcgtacgc tcaggtgcag ctgcaggag 39
<210> 69
15 <211> 42
<212> DNA
<213> artificial sequence
<220>
20 <221> Sequence is completely synthesized
<222> 1-42
<400> 69
gtaaatgtat gggcccttgg tggaggctga agagacggta ac 42
<210> 70
<211> 12
<212> PRT
<213> artificial sequence
<220>
<221> Sequence is completely synthesized
<222> 1-12
35 <400> 70
Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu
1 5 10 12
<210> 71
40 <211> 66
<212> PRT
<213> artificial sequence
<220>
45 <221> Sequence is completely synthesized
<222> 1-66
<400> 71
Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val He Leu Phe Val
50 1 5 10 15
Val He Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala
20 25 30
55 Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys
35 4 0 4 5
Asp Leu Pro Val Leu Asp Gin Leu Leu Glu Gly Gly Ala Ala His
50 55 60
Tyr Ala Leu Leu Pro Gly
65 66
Sequence Listing - 13 -
15:31
GRIFFITH HACK -» IP AUSTRALIA PT
NO. 601 D006
-58-
Claims:
An agonist antibody or fragment thereof which binds to a thrombopoietin receptor, which is selected from the
group consisting of Ab1, Ab2. Ab3. Ab4. Ab5 and Ab6, wherein each Ab1-Ab6 comprises a VH and VL chain,
each VH and VL chain comprising CDR amino acid sequences designated COR1, C0R2 and CDR3
separated by framework amino acid sequences, the amino acid sequences of each CDR in each VH and VL
chain.of AM-Ab6 selected according to the following table:
! Table 1
Abl: VH COR\ VHCDR2 VH CDR3
DNA (SEQIDNO:I) (SEQ ID NO: 3) (SEQ ID NO: 5)
protein (SEQ ID NO: 2) (SEQ ID NO: 4) (SEQ ID NO. 6)
DNA
protein
VL CDR1
(SEQ ID NO: 7)
(SEQ ID NO: 8)
VL CDR2
(SEQ ID NO: 9)
(SEQ ID NO: 10)
VL CDR3
(SEQ ID NO: 1 1)
(SEQ ID NO: 12)
Ab2:
DNA
protein
VH CDR - 1
(SEQ ID NO: 13)
(SEQ ID NO: 14)
VH CDR2
(SEQ ID NO: IS)
(SEQ ID NO: 16)
VHCDR3
(SEQ ID NO: 17)
(SEQ ID NO: 18)
DNA
protein
V iCDRI
(SEQ ID NO: 19)
(SEQ ID NO: 20)
VL CDR2
(SEQ ID NO: 21)
(SEQ ID NO: 22.)
VLCDR3
(SEQ ID NO: 23)
(SEQ ID NO: 24)
Ab3:
DNA
protein
(SEQ ID NO: 25)
(SEQ ID NO: 26)
VH CDR2
(SEQ ID NO: 27)
(SEQ ID NO: 28)
VH CDR3
(SEQ ID NO: 29)
(SEQ ID NO 30)
DNA
protein
VL CDRI
(SEQ ID NO: 19)
(SEQ ID NO: 20)
vt CDR2
(SEQ ID NO: 21)
(SEQ ID NO: 22)
VLCDR3
(SEQ ID NO: 23)
(SEQ ID NO: 24)
AM:
DNA
protein
vhcdri
(SEQ ID NO: 25)
(SEQ ID NO: 26)
,' VH 00552
(SEQ ID NO: 31)
(SEQ ID NO: 32)
VH CDR3
(SEQ ID NO; 33)
(SEQ ID NO: 34)
DNA
protein
VLCDRI
(SEQ ID NO: 35)
(SEQ ID NO: 20)
VL CDR2
(SEQ ID NO: 21)
(SEQ ID NO: 22)
VLCDR3
(SEQ ID NO: 23)
(SEQ ID NO: 24)
At>5:
DNA
protein
VXCDRl
(SEQ ID NO: 3«)
(SEQ ID NO: 37)
VH CDR2
(SEQ ID NO: 38)
(SEQ ID NO: 39)
VH CDR3
(SEQ ID NO: 40)
(SEQ ID NO: 4 1)
05^11/2002 15:31 GRIFFITH HACK ■» IP AUSTRALIA PT
NO. 601 P007
-59-
VUCDR3
(SEQ ID No: 23)
(SEQ ID No; 24)
VH CDR3
(SEQ 10 No: 46)
(SEQ ID No: 47)
VLCDR3
(SEQ ID No: 52)
(SEQ ID No: 53)
15 2. The antibody of Claim 1 which binds to mammalian c-mpl.
3. The antibody of Claim 2, wherein thd antibody stimulates proliferation, differentiation or growth of
megakaryocytes.
2 0 4. Tha antibody of Claim 2, wherein the antibody stimulates megakaryocytes to produce platelets.
5. The antibody of Claim 2. wherein the mammalian c-mpl is human c-mpl.
6. The antibody of Claim 2, which is selected from the group consisting of ScFv, Fab, F(ab') 2 and IgG.
25
7. The antibody of Claim 2. which is a human antibody.
8. The antibody of Claim 2. which is a non-natgrally occurring antibody.
3 0 9. The antibody of Claim 2. which does not stimulate megakaryocytes to produce platelets.
1 0- The antibody of Claim 1 . having a detectable label.
1 1 . The antibody of Claim 1 . which is a monoclonal antibody.
35
12. The antibody of Claim 1 . which is a single chain antibody.
«: \Hai-iR\Kcep',Spi:ci\P374H).cloe 5/'ll.'02
DNA
protein
VL C D R1
(SEQ ID NO: 19)
(SEQ ID NO: 20)
VL CDR2
(SEQ ID NO: 21)
(SEQ ID NO: 22)
10
Ab6:
DNA
protein
DNA
protein
VH CDRI
(SEQ ID NO: 42)
(SEQ (0 NO: 43)
VL CDRi
(SEQ ID NO: 48)
(SEQ ID NO: 49)
VH CDR2
(SEQ ID NO: 44)
(SEQ ID NO: 45)
VL CDR2
(SEQ ID NO: SO)
(SEQ ID NO: 51)
15:31 GRIFFITH HACK -» IP AUSTRAL I Pi PT NO. 601 D008
-60^
1 3. The antibody of Claim 2, which is a mammalian c-mpl binding f ragmerrt-
1 d. An antibody immobilized on an insoluble matrix, wherein the antibody is the antibody of Claim 1 .
5 15. An agonist antibody or fragment thereof which binds to a thrombopoietin receptor, wherein said antibody or
fragment thereof comprises an amino acid sequence which is selected from the group consisting of 1 2E1 0,
12BS, 10F6 and 12D5 as sat forth in Rg. 1 .
1 6. A composition, comprising the antibody or fragment thereof as defined in any one of Claims 1 to 1 5 and a
1 0 pharmaceutically acceptable carrier.
17, The composition of Claim 16, which is sterile.
1 B. The composition of Claim 1 6, whicn Is lyophilized.
15
19. A library of different single chain antibodies, comprising a plurality of the antibody of Claim 1 1 .
20. The library of Claim 1 9, wherein the single chain antibodies are displayed on phage.
2 0 21 . The library of Claim 20, wherein the phage Is M13 and the antibodies are displayed as fusions of coat protein
3.
22. The library of Claim 21 , wherein less than 20% of the phage display more than one fusion on the surface
thereof.
25
30
23. A phage displaying on the surface thereof, the antibody of Claim 1 1 .
24. The phage of Claim 23, wherein the phage is M13 and the antibodies are displayed as fusions of coat protein
3.
25. The phage of Claim 24, wherein the phage displays only one fusion on the surface thereof.
26. A fusion protein, comprising at least a portion of a phage coat protein fused at the amino terminus thereof to
3 5 the antibody of any one of Claims 1 to 1 5.
27. The fusion protein of Claim 26, wherein the phage coat protein is Ml 3 coat protein 3.
05/11/2002 15:31 GRIFFITH HRCK -> IP AUSTRfiL I R PT NO. 601 P009
i
-61-
28. A method of stimulating proliferation, differentiation or growth of megakaryocytes, comprising contacting
megakaryocytes with an effective amount of the antibody of any one of Claims 1 to 15.
5 29. The method of Claim 28, comprising administering the antibody of Claim 1 to a patient in need thereof.
30. A method of increasing platelet production, comprising contacting megakaryocytes with an effective amount
of the antibody of any one of Claims 1 to 15.
10 31 . The method of Claim 30, comprising administering the antibody of Claim 1 to a patient In need thereof.
32. Isolated nucleic acid encoding the antibody of any one of Claims 1 to 15.
33. A vector comprising the nucleic acid of Claim 32.
15
34. A host cell comprising the vector of Claim 33.
35. A method of producing an agonist antibody comprising culturing the cell of Claim 34 under conditions wherein
the nucleic acid is expressed.
20
Dated this 5 th day of November 2002
GENENTECH. INC
By their Patent Attorneys
GRIFFITH HACK
2 5 Fellows Institute of Patent and
Trade Mark Attorneys of Australia
WO 99/10494
PCT/US98/17364
1 /11
VH
F1
CDR1
F2
10F6 1 MAQVQLQESGGEMKKPGESLKISCKGYGYSFATSWIGWVRQMPGRGLEWM
5E5 1 MAEVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWV
10D10 1 MAEVQLVQSGGGWQPGGSLSLSCAVSGITLRTYGMHWVRQAPGKGLEWV
12B5 1 MAQVQLVQSGGGLVRPGGSLSLSCAVSGITLR TYGMHWVRQAPGKGLEWV
12D5 1 MAQVQLVESGGGLVKPGGSLRLSCAASGFTFSSHNMNWVRQAPGKGLEWV
12E10 1 MAQVQLQQSGPGLVKPSETLSLTCTVSGDSISSYYWSWIRQPPGKGLEWI
CDR2
F3
10F6 51 AIMYPGNSDTRHNPSFEDQVTMS ADTSINTAYLQWSSLKASDTAMYYCAR
5E5 51 SYISSSGSTI Y YADS VKGRF T I S RDNSKNTL YLQMNS LRAEDTAVY YC AR
10D10 51 AGISFDGRSEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
12B5 51 AGISFDGRSEYYADSVQGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCAR
12D5 51 S S I S S S S S Y I YYADSVKGRFTI S RDNAKNSL YLQMNSLRAEDTAVYYCAR
12E10 51 GjYIYYSGS-TNYNPSLKSRVTIS VDTSKSQF5LKLSSVTAADTAVYYCAR
10F6
101
5E5
101
10D10
101
12B5
101
12D5
101
12E10
100
CDR3
F4
VL
F1
AGVAGGAFDLWGKGTMVTVSSGGGGSGGGGSGGGGSQSVLTQ-PASVSGS
-WSGEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSTLSAS
-DRGSYGMDVWGRGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSAS
-G-AHYGFDIWGQGTMVTVSSGGGGTGGGGSGGGGSDIQMTQSPSTLSAS
-DRGSTGMDVWGRGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSAS
GRYFDV| WGRGTMVTVSSGGGGSGGGGSGGGGSSYVLTO- PPSVSGS
CDR1
F2
CDR2
10F6 150 PGQSITI SC TGTSSGVGGYNYVS wTYQQHPGKAPKLLI Y GNSNRPS GVPDR
5E5 150 VGDRVAITC RASE GIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSR
10D10 150 IGDRVTITC RASE GIYHWLAWYQQKPGKAPKLLIYECASSLASGAPSR
12B5 149 IGDRVTITC RASE GIYHWLAWYQQKPGKAPKLLIYECASSLAEGAPSR
12D5 150 IGDRVTITC RASE GIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSR
12E10 145 PGQSITI SCTGTSSDVGGYMYVSWYQQHPGKAPKLMIYEGSKRPSGVSNR
F3
CDR3
F4
10F6 2 00 FSASKSGNTASLTISGLQAEDEADYFC STYAPPGIIMFGGGTKLTVL.GAA
5E5 197 FSGSGSGADFTLTISSLQPDDFATYYCQQYSNYPL-TFGGGTKLEVKRAA
10D10 197 FSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPL-TFGGGTKLEILRAA
12B5 196 FSGSGSGTDFTLTISSLQPDDFATYYC QQYSNYPL-TFGGGTELEIKRAA
12D5 197 FSGSGSGTDFTXTISSLQPDDFATYYC QQYSNYPL-IFGGGTKLEIKRAA
12E10 19 5 FSGSKSGNTASLTISGLQAEDEADYYC SSYTTRSTRVFGGGTKLTVLGAA
FIG.. 1
WO 99/10494
PCT/US98/17364
2/11
B CELLS FROM
43 HUMAN DONORS
Purify mRNA
REVERSE TRANSCRIBE
= cDNA
PRIMARY PCR
ScFv
ASSEMBLY
PCR ASSEMBLY
VH 1 VL
> Y \
Gly4 Ser LINKER
VH ^ VL
Sfi ^^; > \ ^-^NOT
CLONING
lacZ P
SIGNAL
LINKER gjV* (hjs)6 AMBER
\ V
6 x 10 9 DIFFERENT CLONES
Y
FIG..2
J
WO 99/10494
PCT/US98/17364
3/11
WO 99/10494
4/11
PCT7US98/17364
ANTIGEN-
COATED
MAXISORP
TUBE
BIND PHAGE
TYPICALLY 3 TO 4
CYCLES (3 TO 5
DAYS)
WASH TUBE
ELUTE PHAGE
WITH
TRIETHYLAMINE
SCRAPE COLONIES
RESCUE WITH
HELPER PHAGE
INFECT E. coli AND PLATE
ON SELECTIVE MEDIUM -
TOOTHPICK COLONIES
INTO 96 WELL
"CELL WELL" PLATE
FIG..4
J
WO 99/10494
PCT/US98/17364
5/11
BIOTINYLATED c-mpl IN SOLUTION
ADD PHAGE TO ANTIGEN, BIND
ADD MAGNETIC BEADS COATED
WITH STREPTAVIDIN
PULL OUT BOUND PHAGE
WITH MAGNET
1
WASH, ELUTE, INFECT
USE DECREASING ANTIGEN CONCENTRATIONS
TO SELECT FOR HIGHEST AFFINITY PHAGE
V v
F/G._5
WO 99/10494
PCT/US98/17364
6/11
MASTER PLATE
REPLICA PLATE TO
2YT, GLUCOSE, CARB
GROW TO MID-LOG
ADD HELPER PHAGE
ON GROWTH
i
PELLET CELLS
CHANGE MEDIA TO
IPTG, CARB
PELLET CELLS, REMOVE
SUPER WITH PHAGE TO
PHAGE ELISA
ON GROWTH
PELLET CELLS, REMOVE
SOLUBLE scFv TO ELISA
ANTI-gp8-HRP
ANTI-c-myc-HRP
scFv
scFv
c-myc TAG
Ag-COATED WELLS
ANTl-c-myc-
COATED WELLS
FIG.. 6
J
WO 99/10494
PCT/US98/17364
7/11
VH
SIGNAL
WW.
VL
LINKER
GENE3
m
B
V
PCR MIX
CONTAINING PRIMERS
A AND B
ADD TINY AMOUNT OF
E. coli FROM COLONIES
1 Kb BAND IN SEEN ON AGAROSE GEL
DIGEST WITH RESTRICTION ENZYME BstNI
ANALYZE PRODUCTS ON 3% AGAROSE GEL
F/G._7
WO 99/10494
8/11
PCT7US98/17364
cm ifl n oo 10 cd o^-i-cvj-r-coio^injo
SmmcQQOiuuiiLU-<<<< CQO
oo -r- co
O OX
m in m
FIG.-8A
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C3>0>0>C3>03C3>a>0>0)0>0>
<<<CQ0QCQOO
oooooooo
FIG. SB
CM O 1-
<<<C0CQCQCQO
cocococococococo
OOOQQLUUJii.
cocococococococo
WO 99/10494
PCT/US98/17364
9/11
CM 00
f c m t- r>-
" o cm co o
O lt> o o
<
J
l.\\\\\\\N\\V\\\\X\XVx\N
Knxxx\\\n\xxx\\xvxxx\x
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r
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CM O CO CD
o o
LU
o
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CD c
DC O
CO
<
X
LU
>
LU
CM
Q
CM
m
CM
CO
o
Q
o
<
CT>
CO
<
CO
Ol
LO
o
CM
O
o
o
WO 99/10494
PCT/US98/17364
10/11
HU-03
(% TOTAL
BOUND)
100.00
75.00 -
50.00 -
25.00 -
0.00
m tpo
0 ScFv
□ FAB
S FAB' 2 ZIPPER
□ FULL LENGTH IgG
1000 100 10
HU-03
(% TOTAL
BOUND)
100.00- 12D5
75.00
50.00 -
25.00 -
0.00
ll
1
1000 100 10
ill
in
m tpo
0 ScFv
□ FAB
S FAB' 2 ZIPPER
□ FULL LENGTH IgG
FIG.. 10B
0.1
HU-03
(% TOTAL
BOUND)
100.00
75.00
BB TPO
0 ScFv
Ll FAB
E FAB' 2 ZIPPER
□ FULL LENGTH IgG
FIG.. IOC
1000
WO 99/10494
PCT/US98/17364
11 / 11
100.00- 12B5
75.00 -I
PLATELETS
(% TOTAL
BOUND) 50.00
25.00
0.00
J
I
9 S
1 1
•I
m tpo
0 ScFv
El FAB
S FAB' 2 ZIPPER
□ FULL LENGTH IgG
F/G._ 1 0D
1 000 1 00 10
0.1
100.00-
75.00-
PLATELETS
(% TOTAL cn nn
BOUND) 50.00
25.00 -
0.00
12D5
1«
I-
Z
r
In
1^
1000 100
10
J
1
j
:1
Is
It
0.1
B TPO
0 ScFv
□ FAB
FAB' 2 ZIPPER
□ FULL LENGTH IgG
FIG.. 10E
100.00-
75.00 -
PLATELETS
(% TOTAL
BOUND) 50.00
25.00 -
0.00
12E10
EE
1
I
M
Pi
1000 100 10
1 1
1$
I
H TPO
0 ScFv
□ FAB
E FAB' 2 ZIPPER
□ FULL LENGTH IgG
FIG.. 10F
0.1
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